Abstract:

An oligosaccharide useful for a Meningitidis A vaccine contains a first
mannose unit having a spacer in the alpha configuration at C-1, which
spacer is capable of conjugating to a protein, and which is connected to
a second mannose unit through a 1,6-linkage which connects C-6 of the
first unit to C-1 of the second unit, wherein the 1,6-linkage comprises a
phosphonate. Related methods of making such compounds, analogous
compounds, or intermediates thereof are also disclosed.

Claims:

1. An oligosaccharide comprising a first mannose unit and a second mannose
unit, wherein the first mannose unit comprises a spacer moiety in the
alpha configuration at C-1, which spacer is capable of conjugating to a
protein,wherein the first mannose unit is connected to the second mannose
unit through a 1,6-linkage which connects C-6 of the first unit to C-1 of
the second unit,and wherein the 1,6-linkage comprises a phosphonate.

2. The oligosaccharide of claim 1, wherein the 1,6-linkage is in the alpha
configuration.

3. The oligosaccharide of claim 1 or 2, wherein the first mannose unit is
a 2-deoxy-2-aza substituted mannose derivative.

4. The oligosaccharide of claim 1 or 2, wherein the second mannose unit is
a 2-deoxy-2-aza substituted mannose derivative.

5. The oligosaccharide of claim 1 or 2, comprising at least three mannose
units.

6. The oligosaccharide of claim 1 or 2, wherein the 1,6-linkage is of the
form [C-1 of second mannose unit] --CH2--P--O--[C-6 of first mannose
unit].

7. The oligosaccharide of claim 1 or 2, wherein each 2-aza substituent
present is selected from NH2, NHAc, and N.sub.3.

8. The oligosaccharide of claim 1, wherein a third mannose unit is
connected to the second mannose unit by a linkage which comprises
phosphorus, and wherein the linkage connects C-6 of the second mannose
unit to C-1 of the third mannose unit.

9. The oligosaccharide of claim 8, wherein the linkage that connects C-6
of the second mannose unit to C-1 of the third mannose unit comprises a
phosphonate.

10. The oligosaccharide of claim 8 or 9, which is of the formula:
##STR00040## wherein each Az is independently selected from NH2,
NHAc, and N3;Z represents the spacer moiety that is capable of
conjugating to a protein, and that may be in protected form or
unprotected or that may be conjugated to a protein;each R1 is
independently H, optionally substituted C1-C6 alkyl, or M, where M
represents a cation;X is O or CH2;each R3 and R4 is
independently selected from the group consisting of H, Ac, Bn, and other
protecting group;and R6 is H, or a protecting group, or a phosphate,
or a linkage to an additional saccharide unit.

11. The oligosaccharide of claim 1 or 8, further comprising a protein that
is conjugated to the oligosaccharide through the spacer moiety that is in
the alpha-configuration at C-1 of the first mannose unit.

13. The oligosaccharide of claim 1 or 8, wherein the spacer moiety
comprises a hydroxyl or an amine, either of which is optionally protected
or is optionally conjugated to a protein.

14. The oligosaccharide of claim 10, wherein R3 is an acyl group and
R4 is H.

15. The oligosaccharide of claim 1, which comprises the formula:
##STR00041## wherein each Az represents an aza substituent;each R3
and R4 independently represents H or a protecting group;R6
represents H, a protecting group, or a linker attached to another
saccharide unit;one of W and X is O, and the other of W and X is
CN2;n is 1 or 2;Yn is OR when n is 1, and when n is 2, one Y is
═O and the other Y is OR,wherein R is H, C1-C6 alkyl, or C6-C12 aryl,
or C6-C12 arylalkyl, or R is M, where M is a cation; andZ is OR', SR', or
NR'2, where each R' is independently H or an optionally substituted
alkyl, acyl, aryl, arylalkyl, heteroalkyl, heteroacyl, heteroaryl, or
heteroarylalkyl group;or Z represents a linker attached to another
saccharide unit or the spacer moiety conjugated to a protein.

16. The oligosaccharide of claim 15, wherein a protein is conjugated to
the oligosaccharide through an amide or ester linkage.

17. The oligosaccharide of claim 15, wherein W is CH2, X is O, Az is
NHAc and n is 2.

18. The oligosaccharide of claim 15 or 16, wherein R is M and Z comprises
--O--(CH2)n--NH--, wherein n is 2-6.

19. The oligosaccharide of claim 15 or 16, wherein each R3 and
R4 is independently H or Ac.

20. A method to make an oligosaccharide, which method comprises:linking a
first moiety which comprises at least one aza substituted mannose unit
through a 1,6-linkage which comprises a phosphonate to a second moiety
which comprises at least one aza substituted mannose unit,wherein the
first moiety comprises a spacer moiety, which spacer moiety is linked to
C-1 of a mannose unit in the alpha-configuration.

21. The method of claim 20, wherein a Mitsunobu reaction is used to link
C-6 of a mannose unit of the first moiety to C-1 of a mannose unit of the
second moiety.

22. The method of any of claims 20-21, wherein the 1,6-linkage is a
1,6-alpha linkage.

23. The method of any of claims 20-21, wherein the linked N-substituted
mannose units comprise the formula (1) ##STR00042## wherein each Az
represents an aza substituent;each R3 and R4 independently
represents H or a protecting group;R6 represents H, a protecting
group, or a linker attached to another saccharide unit; one of W and X is
O, and the other of W and X is CH2;n is 1 or 2;Yn is OR when n
is 1, and when n is 2, one Y is ═O and the other Y is OR,wherein R is
H, C1-C6 alkyl, or C6-C12 aryl, or C6-C12 arylalkyl, or R is M, where M
is a monovalent cation; andZ represents a moiety capable of being
conjugated to a protein, which may be in protected form.

24. The method of claim 23, wherein X is CH2 and W is O.

25. The method of claim 23, wherein at least one aza substituent on a
mannose unit is an amine or substituted amine that is obtained by
reduction of an azide (N3) substituent.

26. The method of claim 25, wherein the amine or substituted amine is at
position 2 on a mannose unit.

27. The method of claim 25, wherein Z or the spacer moiety at the anomeric
center comprises an amine-substituted alkoxy group.

28. The method of claim 23, further comprising linking the second mannose
unit to an additional saccharide by forming a bond between the oxygen of
OR6 in formula (1) and the additional saccharide.

29. The method of claim 28, wherein the additional saccharide comprises at
least one mannose unit.

30. The method of claim 29, wherein the additional saccharide is linked to
the second mannose unit through a 1,6-alpha linkage.

31. The method of claim 28, wherein R6 is H or Ac and the anomeric center
of each mannose unit present is in the alpha configuration.

32. A method for synthesizing an oligosaccharide of alpha-linked mannose
units, said method comprising:combining a mannose unit comprising formula
(2), wherein R6 is C1-C6 acyl or H, and R1, R3, R4,
Az and Z are as defined in claim 15; ##STR00043## with an elongating
monomer of formula (3), wherein Rx represents a C1-C6 acyl group and
M represents H or a cation; ##STR00044## under Mitsunobu reaction
conditions, whereby an oligosaccharide comprising at least two 2-aza
substituted mannose units connected by a 1,6-alpha linkage is obtained.

33. The method of claim 32, wherein the Mitsunobu reaction conditions are
maintained for a prolonged period of time, whereby an oligosaccharide of
formula (4), ##STR00045## wherein p is an integer from 1 to 20 is
obtained.

34. The method of claim 32 or 33, wherein each Az represents NHAc or
N.sub.3.

35. The method of claim 34, wherein p is 1-5

36. The method of claim 34, wherein p is 2-10.

37. The method of claim 32, further comprising conjugating the
oligosaccharide to a protein.

43. A pharmaceutical composition comprising at least one oligosaccharide
of claim 1, and at least one pharmaceutically acceptable excipient.

44. An immunogenic composition comprising at least one compound of claim
1.

45. A Meningitidis A vaccine comprising at least one compound of claim 1.

46. The Meningitidis A vaccine of claim 45, comprising an oligosaccharide
conjugated to a protein.

47. A method of making a mannose derivative useful for the preparation of
an immunogenic oligosaccharide, said method comprising: cyclizing a
compound of formula (2) with an electrophile ##STR00046## to form a
compound of formula (3), ##STR00047## wherein each of R2, R3,
R4 and R6 is independently H or a protecting group;and El
represents a residue derived from an electrophile.

48. The method of claim 47, further comprising the step of replacing El
with a phosphorus group.

49. The method of claim 47, further comprising replacing OR2 in
formula (2) or formula (3) with an azide.

51. The method of claim 50, wherein the Mitsunobu reaction uses a
phosphoryl azide as the azide source.

52. An improved method to prepare a 2-azido-2-deoxy-D-mannopyranose, which
method comprises:forming a triflate at position 2 of a
1,3,4,6-tetra-O-acyl glucopyranose derivative; anddisplacing the triflate
with an azide nucleophile;wherein the improvement comprises minimizing
exposure of the crude reaction mixture to moisture during the steps of
isolation and purification of the product.

53. An immunogenic composition capable of eliciting protective antibodies
against Meningitidis A, said composition comprising an oligosaccharide
having at least two saccharide units covalently attached to each other
through a stabilized phosphorous-containing linkage.

54. The composition of claim 53, wherein the oligosaccharide comprises at
least two mannose units covalently attached to each other through a
stabilized phosphorous-containing linkage.

55. The composition of any of claims 53-54, wherein the oligosaccharide is
conjugated to a protein.

56. The composition of claim 55, wherein the protein is not albumin.

57. The composition of claim 53, comprising at least 2 different
oligosaccharide moieties.

58. The composition of claim 53, wherein said stabilized
phosphorous-containing linkage is a phosphonate.

59. The composition of claim 58, wherein the phosphonate linkage comprises
a 1,6-linkage, formed by a Mitsunobu reaction.

60. The composition of claim 57, comprising at least two different
oligosaccharides that are specific for at least two meningococcal
immunotypes.

61. The composition of claim 53, further comprising a Streptococcus
Pneumoniae antigen.

63. The composition of claim 62, wherein said polysaccharide is conjugated
to a protein.

64. The composition of claim 53, further comprising (an) antigen(s)
derived from at least one of Meningitidis serotypes A, B, C, W135, and Y.

65. The composition of claim 64, wherein said antigen is derived from
Meningitidis serotype C, W135, or Y.

66. The composition of claim 53, further comprising an adjuvant.

67. The composition of claim 66, wherein said adjuvant is Alum.

68. The composition of claim 63, wherein said oligosaccharide is
conjugated to said protein through a bifunctional reagent comprising a
dicarboxylic acid or a derivative thereof.

69. The composition of claim 68, wherein the dicarboxylic acid comprises
adipic acid or suberic acid or a derivative thereof.

70. The composition of claim 68, wherein the bifunctional reagent
comprises a squarate.

71. The composition of claim 53, wherein at least one mannose unit
comprises an alpha-linked spacer moiety at its anomeric center.

Description:

RELATED APPLICATION

[0001]This application claims benefit of U.S. application Ser. No.
60/678,289 filed May 6, 2005, which is incorporated herein by reference
in its entirety.

TECHNICAL FIELD

[0002]The invention relates to a compound useful for a Meningitidis A
vaccine. More particularly, the compound is an oligosaccharide that
comprises a stabilized phosphorous-containing linkage, preferably a
phosphonate linkage. The compound more preferably contains mannose units
and may also contain a spacer in the alpha configuration at C-1 of the
mannose unit. The invention also includes methods of making the
oligosaccharide and improved methods of making mannose-containing
compounds and intermediates.

BACKGROUND ART

[0003]Meningitis is an infection of the meninges, the thin lining that
surrounds the brain and spinal cord. Several kinds of bacteria can cause
meningitis, and N. meningitidis is one of the most important. Others are
Streptococcus pneumoniae and Haemophilus influenzae type b. There are
several subgroups of N. meningitidis, which are differentiated by the
structure of the capsular polysaccharide that surrounds the bacterium.

[0004]Meningitis is caused by both viruses and bacteria. The two major
types of bacteria causing meningitis are Haemophilus influenzae and
Neisseria meningitidis. In the case of H. influenzae only one serotype,
type b, is important, whereas with N. meningitidis twelve serogroups have
been identified, of which groups A, B, C, and W135 are known to cause
epidemics. The various serotypes have different geographical prevalence,
e.g., type B and C are dominant in Europe and North America and Type A in
Africa and South America. The serotyping is based on the structure and
antigenicity of the capsular polysaccharide (CPS) surrounding the
bacteria, and the CPS can also be used as a vaccine against the bacteria.
Especially efficient vaccines (glycoconjugate vaccines) can be made by
attaching the saccharide to a carrier protein. See Plotkin, S. A., and
Orenstein, W. A., Vaccines, 4th ed., Saunders, pages 959-987 (2004),
which is incorporated herein by reference. These glycoconjugates induce a
T-cell dependent immune response with memory and effect also in small
children, while the non-conjugated CPS generally fails to provide either
a memory effect in adults or any substantial immunogenic effect in
infants. The development of type A vaccines has been considered
especially difficult, due to the inherent instability of the anomeric
phosphate diester linkages that are part of the CPS. The repeating unit
of type A is a monosaccharide,
2-acetamido-2-deoxy-α-D-mannopyranose linked 1→6 via a
phosphodiester bridge (FIG. 1). In the native polysaccharide the 3-OH is
acetylated to an extent of about 80%. The immunological importance of
this acetylation has not been completely investigated, but there are
indications that it is not of major significance.

[0005]Neisseria meningitidis serogroup A causes epidemic outbreaks of
meningitis, mainly in parts of Africa south of the Sahara in the
so-called meningitis belt. In the meningitis belt the estimated incidence
for the period 1970-1992 was about 800,000 cases. See Plotkin, S. A., and
Orenstein, W. A., Vaccines, 4th ed., Saunders, pages 959-987 (2004).
The epidemic outbreaks of meningitis are devastating for the region, so
an effective vaccine is urgently needed. The hope is of course, that the
development of a good vaccine, in combination with efforts similar to the
ones against smallpox performed by WHO in the 1960's and 70's could
likewise eliminate meningitis caused by N. meningitides serotype A.
Vaccinations are a much more cost effective way of controlling a disease
than treatment with antibiotics and other therapies, and cost is
especially important in the developing world.

[0006]Vaccines prepared from the polysaccharide coating on the bacterium,
its capsular polysaccharide, are effective in adults. Exposure to this
polysaccharide causes adults to develop an immunogenic response that
protects against meningitis caused by N. meningitidis. A big limitation
with such vaccines, though, is that the immune system of children under
around two years of age does not respond to most polysaccharide antigens.
Unfortunately, this is the age group at greatest risk for bacterial
meningitis. Thus, the polysaccharide vaccines are of no use in young
children. Furthermore, even in older children and adults, these vaccines
induce only short-term immunity. Protection decreases rapidly and is
generally gone by around two years after vaccination.

[0007]Polysaccharides like the N. meningitidis CPS are T-cell independent
antigens, which means that they can give an immune response without the
involvement of T-cells (thymus-derived cells). This response lacks
several important properties that characterize the T-cell dependent
immune response, such as immunological memory, class-switch from IgM to
IgG, and affinity maturation. If the polysaccharide part is connected to
a carrier protein, however, it triggers a cellular immune response that
creates memory effect, and also gives protection in young children. Such
polysaccharides linked to carrier proteins are often referred to as
glycoconjugates, and are especially valuable as vaccines.

[0008]Glycoconjugate vaccines are so called because their production
involves the conjugation of a polysaccharide antigen or other glycosidic
antigen to a carrier protein. The saccharide moiety in glycoconjugate
vaccines is usually a functionalized bacterial CPS, but it can also be
synthetic. Synthetic carbohydrate structures have a number of potential
advantages over those based on carbohydrates from natural sources.
Naturally derived carbohydrates are heterogeneous mixtures and may
include small amounts of natural impurities and contaminants. In
contrast, synthetic carbohydrates can be produced as homogeneous single
compounds in a controlled manner, with little or no batch-to-batch
variability. Another advantage of synthetic structures is that they can
be made to include functional groups for derivitization or modifications
of the carbohydrate moiety that are difficult or impossible to perform on
the native material. The carrier protein is an important factor in the
modulation of the immunogenicity. Various carriers have been used for
conjugation, and the best results have been achieved using detoxified
versions of strongly immunogenic proteins like diphtheria and tetanus
toxins, which have been approved for use in humans. See U.S. Pat. No.
4,354,170. It has also been shown that the immune system reacts more
effectively when patients have already been immunized with the particular
carrier protein.

[0009]A glycoconjugate vaccine is usually made by conjugating the native
capsular polysaccharide structure of the bacterium to a suitable carrier
protein. However there have been problems with that approach due to the
properties of the polysaccharide that encapsulates N. meningitidis. Its
phosphodiester linkage can degrade under the conditions necessary for
attachment of the polysaccharide to proteins, and even after preparation,
glycoconjugates of the native CPS tend to degrade during storage.

[0010]Phosphodiesters are normally quite stabile, but in the capsular
polysaccharide of N. meningitidis, the phosphodiester is linked to the
anomeric center of a carbohydrate residue. Thus one oxygen of the
phosphodiester is also part of an acetal linkage, which makes it
susceptible to hydrolytic cleavage catalyzed by electrophiles such as
acid or metal ions. Cleavage of this bond breaks the polysaccharide down
into smaller pieces. Unfortunately, during the manipulations required to
form a glycoconjugate, or even in a vaccine formulation, the CPS of N.
meningitidis A is subject to such degradation, rendering it difficult to
make and store effective vaccines comprising this particular CPS.

[0011]One way of making the phosphodiester linkage more stable is to
eliminate the oxygen between the phosphorus and the anomeric oxygen, so
that portion of the linkage is no longer susceptible to cleavage by
electrophilic hydrolysis. The exocyclic oxygen at the anomeric center can
be replaced with an isosteric carbon atom, (CH2), transforming the
phosphodiester into its C-phosphonate analogue. This should produce a
stabilized version of the antigenic polysaccharide. One investigation
directed toward this approach has recently been published.
Torres-Sanchez, M. I., et al., Synlett (2005) 7:1147-1151. However, the
authors did not assess the activity of their compounds or disclose an
oligomer of more than two mannose units. Furthermore, their synthesis
approach provided only the beta anomer at the position where the
oligosaccharide is intended to link to a protein, while the native CPS of
N. meningitidis only contains alpha-linkages. Thus there remains a need
for alpha-linked glycoconjugates having stabilized linkages between the
mannose units and for methods to synthesize them.

[0012]Another approach is to stabilize the phosphodiester linkages using
inductive effects from nearby substituents to reduce the electron density
at the anomeric oxygens; this, too, should slow the anticipated
electrophilic degradation mechanisms. The electron-attracting property of
the substituents can be maintained while an oligosaccharide is
constructed, and perhaps also while it is conjugated to a protein, then
removed once the molecule no longer needs to be exposed to destructive
conditions.

[0013]The present invention includes each of these approaches as well as
combinations thereof. One aspect of the invention thus provides an
efficient synthesis of a C-phosphonate analogue of an oligomeric form of
the repeating unit of the N. meningitidis type A capsular polysaccharide,
needed for vaccine development. See Bundle, D. R., et al., J. Biol. Chem.
(1974) 249:2275-2281. The invention particularly provides methods for
introducing a spacer moiety through which the oligosaccharides of the
invention can be conjugated to a protein to make a glycoconjugate
vaccine, and it orients the spacer moiety in the alpha anomeric
configuration. FIG. 2 illustrates a compound having the desired alpha
configuration at this center and one having the beta configuration. Since
the natural CPS of the target organism is a fully alpha-linked oligomer
of mannose units, it is especially desirable to provide the same
alpha-linked configuration in a synthetic immunogen. Even if all of the
linkages between mannose units in a synthetic oligomer portion of a
glycoconjugate are in the alpha configuration, the configuration at the
center through which the oligosaccharide is linked to the protein can be
particularly influential on the immunogenic effects when the
oligosaccharide portion of the glycoconjugate contains fewer than about
10 mannose units.

[0014]The present invention thus provides such compounds and methods for
the preparation of these compounds. The crucial coupling step is often
performed using a C-phosphonate mono-ester and Mitsunobu conditions.
While it may be impractical to prepare polysaccharides rivaling the
natural CPS in size, that is not necessary: even oligosaccharides only a
few mannose-units in length can elicit an immune response. And synthetic
oligosaccharides offer the advantages of selective linkage mechanisms and
increased stability in vivo, each of which should enhance their
immunogenic effectiveness in vivo.

[0015]Another aspect of the invention provides methods to stabilize the
mannose units so that a phosphodiester linkage, the linkage present in
the native capsular polysaccharide, can be used. It includes compounds
and methods that provide stabilization to facilitate preparation of the
protein conjugates of the oligosaccharides. The stabilization is provided
by an electron-attracting group, an azide, at C-2 of at least one of the
mannose units in a polysaccharide, which increases the polysaccharide's
stability so that it can be conjugated to a protein. After conjugation,
the stabilization is less important, and at that point the azide is
typically reduced to an amine and acylated, which provides the
2-acetylamino group that is part of the recurring mannose unit in the N.
meningitidis A capsular polysaccharide.

[0016]Where appropriate, it is also possible to mix the two stabilization
methods; thus an oligosaccharide of the invention may include
azide-containing mannose units that are linked by a phosphodiester at the
adjacent anomeric center in combination with phosphonate-linked mannose
units. This combination may provide additional stabilization, depending
on the orientation of the phosphonate; or it may provide synthetic
advantages such as increased yields and it may avoid complications that
the acetylamine substituent can cause.

[0017]In other aspects, the invention provides methods to synthesize key
precursors for these compounds, pharmaceutical compositions containing
them, and methods to use them to manufacture a medicament. The compounds,
compositions and medicaments may be administered to a subject to induce
an immunogenic response in the subject, which is typically a human.

DISCLOSURE OF THE INVENTION

[0018]The invention provides immunogenic compounds that comprise a
polysaccharide or oligosaccharide and methods to make these compounds, as
well as methods to use them as vaccines to provide protection against
infection by the Neisseria meningitidis A bacterium. The methods allow
the preparation of oligosaccharides comprising at least two mannose units
that are connected through a 1,6-linkage. This linkage is often a
1,6-alpha linkage, and the mannose units are sometimes N-substituted
mannose groups such as those having 2-NHAc or 2-N3 on one or more of
the mannose units. The linkage may take various forms, but often it
comprises a phosphonate or a phosphate. In some embodiments, the
oligosaccharide comprises at least three mannose units linked in
sequence, each of which is typically N-substituted. While the compounds
may contain any number of mannose units, in preferred embodiments the
oligosaccharide portion of the molecule has a molecular weight under
2000. Thus preferably the compounds of the invention include about two to
ten mannose units linked in series, preferably about three to seven.

[0019]The compounds of the invention are adapted to be conjugated to a
protein, since the glycoconjugate forms are much more effective
immunogens for vaccination purposes: they elicit immunogenic responses in
infants, and they elicit cellular responses that provide a memory effect
to prolong the effectiveness of the vaccination. The compounds thus
comprise a spacer moiety that is attached to the first mannose unit in a
chain of mannose units. The spacer unit is specifically designed to
provide a means for conjugation of the oligosaccharide to a protein, or
to provide a means for capping the terminal saccharide unit, such that it
is unreactive, for example to further chain elongation/modification
reactions. Typically, this spacer moiety possesses an amine, carboxylate,
or hydroxyl group for coupling to a complementary group on a protein
carrier, but other groups known in the art to provide a way to conjugate
an oligosaccharide to a protein are also included. Alternatively, the
spacer moiety possesses a protecting or capping group, such as an alkyl,
aryl, or acyl group, as well as others well know in the art and/or
disclosed herein. In compounds of the invention, this spacer moiety is in
the alpha configuration so that it most closely resembles the linkages in
the natural CPS of N. meningitidis and does not interfere with the
desired immunogenic effect, even in short oligosaccharides of the
invention.

[0020]The compounds of the invention often comprise the structure shown in
formula (1):

[0029]or Z represents a linker attached to another saccharide unit or a
spacer moiety conjugated to a protein.

[0030]Often in these compounds, X is O and W is CH2, and Az is often
N3, NH2 or NHAc; and often n is 2

[0031]The compounds of the invention are synthesized in part by art-known
methods, but a number of novel methods for forming these molecules are
also part of the invention. For example, the 1,6-alpha linkage in a
compound of formula (1) can be constructed with a Mitsunobu reaction as
described herein. Also, the N-substituents such as Az can be inserted as
an azide by methods provided herein, and can then be reduced to provide
an amine or substituted amine, which is often at position 2 of a mannose
unit, as shown in formula (1). Typical methods for reduction of the azide
include catalytic hydrogenation and borohydride reduction, typically
using catalytic amounts of a nickel salt.

[0032]The invention also provides a method to make phosphonate-substituted
mannose units for assembling the oligosaccharides described herein. For
example, a compound of formula (2), which is available from glucose, can
be converted into a precursor for a phosphonate ester useful for forming
the phosphonate-comprising 1,6-alpha-linkage shown in formula (1) where W
is CH2 and X is O. The method includes cyclizing a compound of
formula (2) with an electrophile

##STR00002##

[0033]to form a compound of formula (3)

##STR00003##

[0034]wherein each of R2, R3, R4 and R6 is
independently H or a protecting group;

[0035]and El represents the residue of electrophile.

[0036]The residue of an electrophile, El, is the portion of the
electrophilic reagent used to initiate cyclization that remains attached
to the carbon. El can then be replaced, either directly or indirectly, by
a phosphorus-containing groups such as a phosphonate group by the methods
described herein. This provides a glucose derivative rather than an
N-substituted mannose unit, i.e., it has the wrong stereochemistry at
C-2; thus the invention further provides methods to convert OR2 in
formula (2) or formula (3) into an Az group having the proper
stereochemistry by displacing an activated form of the oxygen of OR2
with an azide. The method comprises a Mitsunobu reaction, which typically
uses a phosphoryl azide as the azide source.

[0037]The invention also provides a method to prepare a
2-azido-2-deoxy-D-mannopyranose, which method comprises forming a
triflate at position 2 of a 1,3,4,6-tetra-protected glucopyranose
derivative; and displacing the triflate with an azide nucleophile. In
some embodiments, the 1,3,4,6-tetra-protected glucose derivative is a
1,3,4,6-tetra-O-acyl glucopyranose, which is readily available. See,
e.g., Helferich, B., et al., Ber. Dtsch Chem. Ges. (1962) 95:2604-2611.
1,3,4,6-Tetra-O-acetyl glucopyranose is sometimes preferred.

[0038]The immunogenic activity of compounds and compositions comprising
the oligosaccharides described herein is often enhanced by conjugation of
the oligosaccharide to a protein. Thus in many embodiments, the invention
includes conjugating the oligosaccharide to a protein through a spacer
moiety attached to the C-1 carbon of the first mannose unit in the chain.
In some embodiments, the protein is one chosen for its ability to enhance
the immunogenic response in a human, and the detoxified toxoids from
diphtheria and tetanus are often used. Thus the compounds of the
invention often include a spacer moiety, which may be Z in formula (1) or
may be attached to any of the available hydroxyl groups or N-substituents
on the mannose units, and which facilitates attachment to a protein.
Typically, the connection to the protein is through Z, thus Z is often a
spacer moiety that is capable of being conjugated to a protein. In many
of the compounds of the invention, Z is in a protected form. Suitable
protecting groups depend on the exact nature of Z, and selection and
usage of such protecting groups is within the ordinary skill in the art.
Examples of such protecting groups and details of their usage are
available in, for example, Greene, T. W., and Wuts, P. G. M., Protective
Groups in Organic Synthesis, 2d ed. (1991). Similarly, the invention
provides methods to use Z to connect the oligosaccharide to a protein to
produce an immunogenic composition.

[0039]The invention also provides vaccine compositions comprising an
oligosaccharide of the invention, which are useful for eliciting an
immunogenic response in a mammal. Typically the mammal is a human subject
since N. meningitidis is believed to be pathogenic only in humans, but
eliciting an immune response in other mammals is of value, too, and can
be used to provide immune components such as antibodies. Thus the
invention provides immunogenic compositions and methods to use these to
elicit an immunogenic response in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1 shows the structure of the capsular polysaccharide of N.
meningitidis A.

[0041]FIG. 2 is an illustration of the alpha and beta anomers at the
center through which the oligosaccharide is conjugated to a protein to
enhance its immunogenicity.

[0043]FIG. 4 shows the relationship between the specific antibody
responses induced by synthetic glycoconjugate and the control
oligosaccharide conjugate at different vaccine doses.

MODES OF CARRYING OUT THE INVENTION

[0044]In one aspect, the invention provides an oligosaccharide comprising
a first mannose unit and a second mannose unit, wherein the first mannose
unit comprises a spacer in the alpha configuration at C-1. This spacer is
capable of conjugating to a protein, and the first mannose unit is
connected to the second mannose unit through a 1,6-linkage which connects
C-6 of the first unit to C-1 of the second mannose unit. The 1,6-linkage
comprises a phosphonate in some embodiments. In some embodiments, the
1,6-linkage is in the alpha configuration. In some of these embodiments,
the first mannose unit is a 2-deoxy-2-aza substituted mannose derivative,
and in some of the embodiments the second mannose unit is a 2-deoxy-2-aza
substituted mannose derivative. Certain embodiments have two or three, or
more than three of these 2-aza substituted mannose units.

[0045]In some of the embodiments of the invention, the 1,6-linkage is of
the form [C-1 of second mannose unit] --CH2--P--O-- [C-6 of first
mannose unit], i.e., the phosphonate carbon is connected to C-1 of the
second mannose unit and a phosphonate ester oxygen is bonded to C-6 of
the first mannose unit. These mannose units are optionally protected, and
in many embodiments one or both of these two mannose units comprises a
2-aza substituent which is selected from NH2, NHAc, and N3.
Where these embodiments include a third mannose unit, it is sometimes
connected to the second mannose unit by a linkage which comprises
phosphorus, and wherein the linkage connects C-6 of the second mannose
unit to C-1 of the third mannose unit. This linkage often comprises a
phosphonate, which is often lined to the second mannose unit through a
phosphonate ester linkage, and to the third mannose unit through a P--C
bond of the phosphonate.

[0046]In other aspects, the invention provides an oligosaccharide of
formula (1):

[0049]R6 represents H, a protecting group, or a linker attached to
another saccharide unit;

[0050]one of W and X is O, and the other of W and X is CH2;

[0051]n is 1 or 2;

[0052]Yn is OR when n is 1, and when n is 2, one Y is ═O and the
other Y is OR,

[0053]wherein R is H, C1-C6 alkyl, or C6-C12 aryl, or C6-C12 arylalkyl, or
R is M, where M is a cation; and

[0054]Z is OR', SR', or NR'2, where each R' is independently H or an
optionally substituted alkyl, acyl, aryl, arylalkyl, heteroalkyl,
heteroacyl, heteroaryl, or heteroarylalkyl group;

[0055]or Z represents a linker attached to another saccharide unit or a
spacer moiety conjugated to a protein.

[0056]In some embodiments, compounds that comprise formula (1) are
conjugated to a protein through an amide or ester linkage. Often in
compounds of formula (1), W is CH2 and X is O, and in many of such
embodiments, Az is NHAc and n is 2. Also, in many embodiments of this
aspect, R is M and Z comprises --O--(CH2)n--NH--, wherein n is
2-6. In embodiments of these compounds based on formula (1), each R3
and R4 is independently H or Ac. Often R3 is Ac, and optionally
both R3 and R4 are either H or Ac.

[0057]In other aspects, the invention provides methods to make an
oligosaccharide, which methods comprise linking a first moiety which
comprises at least one aza substituted mannose unit through a 1,6-linkage
which comprises a phosphonate to a second moiety which comprises at least
one aza substituted mannose unit. The first moiety in these embodiments
often comprises a spacer moiety, which spacer moiety is linked to C-1 of
a mannose unit in the alpha-configuration. In some embodiments of these
methods, a Mitsunobu reaction is used to link C-6 of a mannose unit of
the first moiety to C-1 of a mannose unit of the second moiety. In many
of the embodiments of these methods, the 1,6-linkage is a 1,6-alpha
linkage.

[0058]In some of these aspects, the linked aza substituted mannose units
comprise the formula (1)

[0061]R6 represents H, a protecting group, or a linker attached to
another saccharide unit;

[0062]one of W and X is O, and the other of W and X is CH2;

[0063]n is 1 or 2;

[0064]Yn is OR when n is 1, and when n is 2, one Y is ═O and the
other Y is OR,

[0065]wherein R is H, C1-C6 alkyl, or C6-C12 aryl, or C6-C12 arylalkyl, or
R is M, where M is a monovalent cation; and

[0066]Z represents a moiety capable of being conjugated to a protein,
which may be in protected form.

[0067]In some embodiments of the method, in the compounds that comprise
formula (1), X is CH2 and W is O. In these embodiments, often at
least one aza substituent on a mannose unit is an amine or substituted
amine that is obtained by reduction of an azide (N3) substituent. In
preferred embodiments, the amine or substituted amine is at position 2 on
a mannose unit. In some embodiments of these methods, the oligosaccharide
comprises a spacer moiety which may be Z in a compound of formula (1),
which is at the anomeric center of a mannose unit. This spacer unit may
comprise an amine-substituted alkoxy group, which is optionally in
protected form. For example, it may be of the formula
--O--(CH2)n--NH--PG, where PG represents H or a protecting
group; the protecting group is often an alkoxycarbonyl such as methoxy
carbonyl; t-butyloxy carbonyl; or benzyloxycarbonyl. Thus NH--PG is often
a carbamate group.

[0068]In some embodiments of these methods, the method further comprises
linking the second mannose unit to an additional saccharide by forming a
bond between the oxygen of OR6 in formula (1) and the additional
saccharide. This saccharide may be a mannose unit or comprise a mannose
unit, and in many embodiments the additional saccharide is linked to the
second mannose unit through a 1,6-alpha linkage. In these embodiments,
often R6 is H or Ac and the anomeric center of each mannose unit
present is in the alpha configuration to most closely resemble the
natural CPS of N. meningitidis.

[0069]In other aspects, the invention provides an oligosaccharide of the
formula (1'):

##STR00006##

[0070]wherein each Az is independently selected from NH2, NHAc, and
N3;

[0071]Z represents a spacer moiety that is capable of conjugating to a
protein, and that may be in protected form or unprotected form and that
may be conjugated to a protein;

[0074]each R3 and R4 is independently selected from the group
consisting of H, Ac, Bn, and other protecting groups;

[0075]and R6 is H, or a protecting group, or a phosphate, or a
linkage to an additional saccharide unit.

[0076]In this aspect of the invention, some embodiments comprise a protein
that is conjugated to the oligosaccharide through a spacer moiety that is
in the alpha-configuration at C-1 of the first mannose unit. The protein
is sometimes an inactivated bacterial toxin selected from diphtheria
toxoid, pertussis toxoid, E. coli LT, E. coli ST, Pseudomonas aeruginosa
exotoxin (rEPA), or tetanus toxoid, or the protein may be CRM197. The
protein in these embodiments may be linked to the oligosaccharide of
formula (1) through a spacer moiety, which comprises a hydroxyl or an
amine, either of which is optionally protected or is optionally
conjugated to a protein. In some embodiments of this aspect, R3 is
an acyl group and R4 is H. The alternate or preferred embodiments of
formula (1) also apply to formula (1').

[0077]In certain aspects, the invention provides a method to synthesize an
oligosaccharide of alpha-linked mannose units, which comprises combining
a mannose unit comprising formula (2), wherein R6 is C1-C6 acyl or
H, and R1, R3, R4, Az and Z are as defined in claim 15;

##STR00007##

[0078]with an elongating monomer of formula (3), wherein Rx
represents a C1-C6 acyl group and M represents H or a cation;

##STR00008##

[0079]under Mitsunobu reaction conditions, to produce an oligosaccharide
comprising at least two 2-aza substituted mannose units connected by a
1,6-alpha linkage. In some embodiments of this method, the Mitsunobu
conditions include the use of either diisopropyl azodicarboxylate (DIAD)
or diethyl azodicarboxylate (DEAD) and either triphenyl phosphine or a
substituted triphenyl phosphine such as tris(p-chlorophenyl)phosphine. In
some embodiments, DIAD and tris(p-chlorophenyl)phosphine are used, and
triethylamine is used in excess. In certain embodiments, the Mitsunobu
reaction conditions are maintained for a prolonged period of time, and
the product is an oligosaccharide of formula (4),

##STR00009##

[0080]wherein p is an integer from 1 to 20.

[0081]In many of these embodiments, each Az represents either NHAc or
N3. In some of these embodiments, p is 1-10, and in others, p is
about 1-5 or p is 2-4. In certain of these embodiments, the methods of
the invention further include a method to conjugate the oligosaccharide
of formula (4) to a protein. Optionally this is done through Z, which is
often a spacer moiety selected to be capable of conjugating to such
proteins. In some of these embodiments, the protein is an inactivated
bacterial toxin selected from diphtheria toxoid, pertussis toxoid, E.
coli LT, E. coli ST, Pseudomonas aeruginosa exotoxin (rEPA), or tetanus
toxoid. In other embodiments, the protein is CRM197.

[0082]In other aspects, the invention provides an oligosaccharide that is
prepared by the foregoing methods. The oligosaccharide compounds prepared
by these methods are immunogenic compounds, and typically elicit an
immunogenic response in a treated mammal which provides at least partial
immunity to infections caused by N. meningitidis.

[0083]In still other aspects, the invention provides a method to use any
oligosaccharide compounds of the invention to elicit an immunogenic
response, typically by administration to a mammal. In many embodiments,
these compounds are used as a Meningitidis A vaccine component; the
methods thus often comprise administering an effective amount of the
vaccine component to a subject, thereby providing an immunogenic
response. The immunogenic response provides at least partial resistance
or immunity in the subject to meningitis caused by N. meningitidis A.

[0084]The invention also provides pharmaceutical compositions comprising
at least one oligosaccharide of the invention admixed with at least one
pharmaceutically acceptable excipient to provide a pharmaceutical
composition that is immunogenic. In some embodiments, these compositions
are thus vaccines including a Meningitidis A vaccine comprising any
compound of the invention. In many embodiments, the vaccine comprises at
least one oligosaccharide conjugated to a protein.

[0085]In yet other aspects, the invention provides methods for making a
mannose derivative useful for the preparation of an immunogenic
oligosaccharide, including those of the invention. These methods comprise
cyclizing a compound of formula (2) with an electrophile

##STR00010##

[0086]to form a compound of formula (3),

##STR00011##

[0087]wherein each of R2, R3, R4 and R6 is
independently H or a protecting group;

[0088]and El represents a residue derived from an electrophile.

[0089]In certain embodiments, the methods include an additional step that
comprises the step of replacing El with a phosphorus group. The
phosphorus group is typically a phosphonate. In some embodiments, the
invention also provides methods for OR2 in a compound of formula (2)
or formula (3) with an azide. In preferred embodiments, the replacement
of OR2 comprises a Mitsunobu reaction. In some such embodiments, a
phosphoryl azide provides the azide for the Mitsunobu reaction.

[0090]In still other aspects, the invention provides an improved method to
prepare a 2-azido-2-deoxy-D-mannopyranose, which method comprises:

[0091]forming a triflate at position 2 of a 1,3,4,6-tetra-O-acyl
glucopyranose derivative; and

[0092]displacing the triflate with an azide nucleophile.

[0093]Other methods for making such compounds may be used, and the present
improved method provides for precautions to minimize exposure to moisture
during work-up and isolation of the product. This resulted in greatly
improved yields over known methods, e.g., Popelova, et al., Carbohydrate
Res. (2005) 340:161-166. In a preferred embodiment, the
1,3,4,6-tetra-O-acyl glucopyranose derivative is 1,3,4,6-tetra-O-acetyl
glucopyranose, and the product is 2-azido-1,3,4,6-tetra-O-acyl
mannopyranose.

[0094]In other aspects, the invention provides an immunogenic composition
capable of eliciting protective antibodies against Meningitidis A, which
comprises an oligosaccharide having at least two saccharide units, which
are typically mannose units, covalently attached to each other through a
stabilized phosphorous-containing linkage. In many such embodiments, at
least one saccharide unit, which may be a mannose unit, comprises an
alpha-linked spacer moiety at its anomeric center. The stabilized
phosphorus-containing linkage can comprise a phosphonate. In certain
embodiments, these oligosaccharides comprise at least two mannose units
covalently attached to each other through a stabilized
phosphorous-containing linkage. In many of the embodiments, the
oligosaccharide is conjugated to a protein. Many proteins such as those
described above as suitable carrier proteins can be used, but in
preferred embodiments the protein is not albumin. In some embodiments,
the immunogenic compositions of the invention comprise at least 2
different oligosaccharide moieties. In many of the embodiments, the
stabilized phosphorus-containing linkage between two saccharides
comprises a phosphonate. In many such embodiments, the phosphonate
linkage is a 1,6-linkage, formed by a Mitsunobu reaction, and in certain
embodiments, the oligosaccharide of any of the foregoing compositions
comprises at least one mannose unit which comprises an alpha-linked
spacer moiety at its anomeric center.

[0095]In some of the immunogenic compositions of the invention, the
composition comprises at least two different oligosaccharides that are
specific for at least two meningococcal immunotypes. The compositions may
also include other antigenic compounds, and in some embodiments, the
compositions further comprise a Streptococcus Pneumoniae antigen. This
antigen is a polysaccharide in some embodiments, and in many of these
embodiments the polysaccharide is conjugated to a protein.

[0096]In some embodiments, the compositions of the invention also comprise
at least one antigen derived from a Meningitidis of serotypes A, B, C,
W135, or Y. In some preferred embodiments, this antigen is derived from
Meningitidis serotype C, W135, or Y. The compositions of the invention
often further comprise an adjuvant, and in some of the embodiments the
adjuvant is alum.

[0097]In many compositions of the invention, the oligosaccharide component
is conjugated to a protein through a bifunctional reagent comprising a
dicarboxylic acid or a derivative thereof. In many embodiments, this
dicarboxylic acid comprises adipic acid or suberic acid or a derivative
thereof. In other embodiments, the bifunctional reagent comprises a
squarate.

[0098]The preparation of phosphonate-linked compounds within the scope of
the invention can be accomplished by making an acceptor monomer and an
elongating monomer, each of which is an appropriately modified
mannopyranose ring, referred to as a mannose unit. The elongating monomer
is attached to the acceptor through a phosphonate linkage to make a
disaccharide. The elongating monomer can be adapted to allow its C6
hydroxyl to be deprotected selectively; thus, if a longer oligosaccharide
is desired, the elongating monomer in the dimeric saccharide can become
an acceptor. It can be deprotected without deprotecting the C3 and C4
hydroxyls, so the C6 hydroxyl can be linked to the phosphonate of another
elongating monomer. This process can be iterated as many times as
necessary to provide an oligosaccharide of the desired length.

[0099]FIG. 3 depicts how an acceptor monomer and an elongation monomer are
linked to form an oligosaccharide having two mannose units (i.e., a
disaccharide), and how this disaccharide can be extended further by
deprotection of the C6 hydroxyl followed by attachment of another
elongation monomer. Optionally, the acceptor monomer may be attached to a
solid support by a cleavable linker to facilitate handling and isolation.
In that case, the oligosaccharide can remain attached to the solid
support through multiple iterations of the elongation process, and can
eventually be cleaved to release a relatively pure oligosaccharide
product of the desired length. The spacer moiety Z is sometimes used to
link the oligosaccharide to the solid support during synthesis, and then
used, optionally with modification, to conjugate the oligosaccharide to a
protein.

[0100]The invention provides methods to make immunogenic oligosaccharides
containing two or more modified mannose units, as well as longer
oligosaccharide or polysaccharide molecules, and methods to make the
oligo- and/or polysaccharides into more immunogenic materials by
conjugating them to a protein. The mannose units are pyranose rings
having the basic structure of a mannose ring, but having at least some
structural modifications. In many embodiments, the invention includes at
least one substituent where a nitrogen atom or other heteroatom is bonded
to a ring carbon in place of one of the hydroxyl groups of mannose; as
used herein, such substituted rings are considered mannose units as long
as the array of substituents on the pyranose ring has the same relative
stereochemistry as the array of stereocenters on mannose. Note that the
mannose configuration does not define the stereochemistry at C-1; C-1,
which is the anomeric center in a mannose unit, can be in either the
alpha or the beta configuration. Thus a mannose unit can have a C, N, or
S atom attached in place of one of the mannose hydroxyls, or can have two
or more of such modifications. The structure and numbering of carbon
atoms for D-mannose is provided here for reference.

##STR00012##

[0101]The methods of the invention can be used with mannose units having
the absolute stereochemistry of either D-mannose or an L-mannose; in many
embodiments the absolute stereochemistry is that of D-mannose. However,
an oligosaccharide of the invention can include one or more mannose units
having the L-mannose configuration. It can also, of course, be linked to
additional saccharide or peptide moieties, for example, as long as it
includes at least two mannose units linked as described herein.

[0102]The oligosaccharides of the invention include at least two pyranose
rings linked together; if only two rings are included, the molecule can
be described as a dimer; with three linked in sequence it can be
described as a trimer; and so forth. However, the generic term
oligosaccharide as used herein includes these smaller embodiments as well
as longer polymer versions having three or more, five or more, seven or
more, ten or more, fifteen or more, twenty or more, and more than 25
monomers in sequence. Versions having more than about five saccharide
rings are sometimes referred to herein as polysaccharides. In many
embodiments the oligosaccharide comprises at least three mannose units.

[0103]Regardless of the number of mannose units included, it is sometimes
desirable to link the oligosaccharide to other molecular features, so the
mannose units may be substituted with various groups, and the
oligosaccharide may be linked through one or more mannose units,
typically through one of the terminal mannose units, to another moiety.
In some embodiments, it is beneficial to link the oligosaccharide to a
protein, and these glycoconjugate forms are specifically included within
the invention. Typically the glycoconjugate is formed by conjugation of
the protein of interest to a functional group on the spacer moiety
attached at C-1 of the first mannose in the oligosaccharide, and the
spacer moiety is in the alpha configuration. In many embodiments, it is
desirable to attach a protecting group or other functionality to one or
more of the heteroatom substituents on the mannose unit. In some
preferred embodiments, the heteroatom substituents are selected from OH,
OAc, NH2, N3, NHAc, and an O, N, or S that is connected to a
protecting group or to a protein.

[0104]The mannose units are typically connected by a 1,6-linkage. The 1,6
linkage means that the C-1 carbon, the anomeric center, of the second
mannose unit is connected, either directly or indirectly, to the C-6
carbon of the first mannose unit by a linkage that includes no
intervening saccharide rings. Typically the linkage between two mannose
units will include at least two atoms, generally two or more non-carbon
atoms, between the mannose unit carbon atoms. In many embodiments the
linkage is of the general form:

##STR00013##

[0105]where each of D, E and F represents a heteroatom selected from O, N,
S, Si, and P, and each of the tetrahydropyran rings represents the ring
of a mannose unit. In some embodiments E represents a phosphorous atom,
which is often in the oxidation state of a phosphate or a phosphonate.
Typically, at least one of D and F in this formula is oxygen (O), and in
some embodiments both D and F are oxygen. Particular embodiments include
those wherein D-E-F represents O--P(O)--O or C--P(O)--O or O--P(O)--C.
Sometimes D-E-F is preferably a linkage comprising --CH2--P(O)--O--.

[0106]In some embodiments, the linkage between adjacent mannose units is a
1,6-alpha (1,6-α) linkage. For mannose units, the 1,6-alpha linkage
means that the non-H substituent at C-1, which corresponds to the
anomeric center carbon of mannose, is in an `anti` relative
stereochemical relationship to the non-H substituent at C-2.
Alternatively, one or more mannose units in an oligosaccharide may be
linked through a beta linkage. (The configuration having the linkage at
the anomeric center `syn` to the C-2 substituent in a mannose is referred
to as a beta linkage.) However, typically the compounds are linked
together in an alpha relationship, regardless of the nature of the
linkage itself.

[0107]The following structures illustrate the difference between the alpha
and beta linkage orientations.

##STR00014##

[0108]Many embodiments of the invention include an aza substituted mannose
unit, which means that at least one of the mannose hydroxyls has been
replaced with a substituent referred to herein as an "aza" substituent.
This is a substituent wherein at least the atom linked directly to the
mannose unit is nitrogen. Aza substituents include NH2 and various
substituted amines, including alkylated and dialkylated amines, and
acylated and diacylated amines, where the acyl groups include heteroacyls
such as benzyloxycarbonyl and methoxycarbonyl as well as such acyl groups
as acetyl, formyl, and benzoyl. Other embodiments of the aza substituent
include NO2 and N3. Some embodiments of the aza substituted
mannose units of the invention have the aza substituent at C-2 of the
mannose ring, and preferred embodiments include compounds and methods
wherein the aza substituent on a mannose unit is NH2, N3, or
NHAc.

[0109]Thus in some embodiments, the compounds and methods of the invention
comprise a moiety of formula (1):

[0118]or Z represents a linker attached to another saccharide unit or a
spacer moiety conjugated to a protein.

[0119]Preferred aza substituents are those in which a nitrogen atom is
directly bonded to the mannose unit. Examples of such substituents
include N3, NH2, NH-alkyl, NH-acyl, NH-aryl and the like. Each
N in these substituents can optionally include one or two groups selected
from alkyl, aryl, and acyl, where each alkyl, acyl and aryl is optionally
substituted.

[0120]The term "protected" or a "protecting group" with respect to
hydroxyl groups, amine groups, and sulfhydryl groups refers to forms of
these functionalities which are protected from undesirable reaction with
a protecting group known to those skilled in the art such as those set
forth in Protective Groups in Organic Synthesis, which can be added or
removed using the procedures set forth therein. Examples of protected
hydroxyl groups include, but are not limited to, silyl ethers, such as
TBDMS or TBS, such as those obtained by reaction of a hydroxyl group with
a reagent such as, but not limited to, t-butyldimethyl-chlorosilane,
trimethylchlorosilane, triisopropylchlorosilane, and
triethylchlorosilane; substituted methyl and ethyl ethers such as, but
not limited to methoxymethyl ether, methythiomethyl ether,
benzyloxymethyl ether, t-butoxymethyl ether, 2-methoxyethoxymethyl ether,
tetrahydropyranyl ethers, 1-ethoxyethyl ether, allyl ether, and benzyl
ether; esters such as, but not limited to, benzoyl, benzoylformate,
formate, acetate, trichloroacetate, and trifluoracetate. Examples of
protected amine groups include, but are not limited to, benzyl or
dibenzyl, amides such as, formamide, acetamide, trifluoroacetamide, and
benzamide; imides, such as phthalimide, and dithiosuccinimide; and
others. In some embodiments, a protecting group for alcohols is a benzyl
group or an acetyl group, and a typical protecting group for an amine
herein is acetyl or a benzyl or t-butyl carbamate.

[0121]"Protecting group" as used herein include those moieties recognized
in the art as protecting groups for the heteroatoms N, O and S, and are
described, for example, in the reference book by Greene, T. W., and Wuts,
P. G. M., Protective Groups in Organic Synthesis, 2d ed., Wiley and Sons
(1991), which is incorporated herein by reference. Acetyl (Ac) is one
such protecting group, and because the natural CPS of N. meningitides is
often acetylated, particularly at C-3, in some embodiments of the
invention, one or more of the protecting groups is Ac; and in some
preferred embodiments, R3 on at least one of the mannose units
represents Ac.

[0122]In some of these compounds, one of X and W is CH2, while the
other one is O; for example, in some embodiments X and W are both O, and
in others, W is CH2 and X is O. Within a given compound of the
invention that is oligomeric and contains three or more mannose units,
different embodiments of the linkage may be present; thus two mannose
units linked together by a linkage comprising a phosphonate may be linked
to a third mannose unit via a phosphate diester linkage without departing
from the invention.

[0123]It is often desirable to conjugate the oligosaccharides of the
invention with another molecule, typically a protein. Conjugates made by
such methods are within the scope of the invention. Thus conjugation of a
saccharide to a protein is often accomplished by adding a reactive
bi-functional molecule to the protein then exposing the derivatized
protein to the saccharide, so that the saccharide becomes covalently
linked through the linker provided by the bi-functional reagent, which
often is attached to one of the hydroxyl groups of the saccharide. In the
controlled synthesis of compounds of the invention, however, it is
especially convenient to include a spacer moiety specifically designed
for conjugating the oligosaccharide to a protein. Such a spacer moiety
can be attached to any one of the mannose units in an oligosaccharide;
however, typically it is on a terminal mannose unit, either the first
`acceptor` monomer, or the last `elongation` monomer. Frequently it is at
C-1 of the acceptor or C-6 of the elongation monomer, and in compounds of
formula (1) it may be the group represented by Z or the one represented
by R6.

[0124]The spacer moiety for conjugating the oligosaccharide to a protein
may comprise a multi-atom spacer moiety to make the immunogenic epitope
of the oligosaccharide more available and thus more effective.
Alternatively, for example, Z in a compound such as those represented by
formula (1) can be a single atom such as O or N or S, and a space between
the oligosaccharide and the protein may be provided by a bi-functional
reagent used to link the oligosaccharide and a carrier protein together.
The bifunctional reagents suitable for use in the glycoconjugates of the
invention include those known in the art. Examples of such include
di-carboxylic acids such as malonic, succinic, adipic and suberic acids
or activated versions thereof, and squaric acid derivatives. These types
of reagents are particularly convenient for linking a compound wherein
the spacer moiety comprises an amine to a protein.

[0125]In some embodiments, the spacer moiety is at least two or three
atoms in length, though it optionally may be a single atom or it may be
much longer; one example is a compound of formula (1), wherein Z
represents a 2-aminoethoxy group. The 2-aminoethoxy group or a homolog
thereof can be introduced into the acceptor monomer before elongating
monomers are attached, and is optionally introduced in protected form so
it does not participate in the subsequent reactions. The spacer moiety
must include at least one functional group that is capable of being used
to link the oligosaccharide to a protein, and typically it includes a
heteroatom, N, O or S, for this purpose, though other groups such as a
diene or a dienophile for linkage through a Diels-Alder reaction, or a
carboxylate group for conjugating the protein to the oligosaccharide
through an ester or amide are also contemplated. In many embodiments such
as the 2-aminoethoxy group discussed above, the spacer moiety includes a
nitrogen for this purpose; the nitrogen may be protected when it is
introduced and during construction of the oligosaccharide, but is capable
of being selectively deprotected after the oligosaccharide has been
constructed under conditions that do not destroy the oligosaccharide. The
amine of the spacer moiety is then readily acylated or alkylated, for
example, with a bi-functional reagent, the other end of which is
similarly attached to a protein. The order of such attachment, i.e.,
which piece of the glycoconjugate is attached to the bi-functional
reagent first, is unimportant and is determined by the judgment of the
practitioner. Some typical, but non-limiting examples of spacer groups
include -Het-(CH2)n-A, -Het-Ph-A,
-Het-(CH2)n-Ph-(CH2)n-A and substituted forms
thereof, wherein each Het represents a heteroatom, usually O, S or N;
each Ph represents a phenyl group, optionally substituted; and each n
represents an integer from 1-10; A represents a functional group or a
residue thereof that is capable of or links the spacer to the protein,
such as an N, O, or S, or ester, an amide, or other carboxyl-containing
group, a diene, or a dienophile. Preferably, the spacer comprises OR',
SR', or NR'2, where each R' is independently H or an optionally
substituted alkyl, acyl, aryl, arylalkyl, heteroalkyl, heteroacyl,
heteroaryl, or heteroarylalkyl group and may further comprise A.

[0126]M in formula (1) can represent an alkali metal cation, selected from
Li, Na, K, and Cs, or an ammonium salt NR4.sup.+, where each R is
independently H or C1-C6 alkyl or C1-C6 heteroalkyl. Alternatively, it
can represent any other pharmaceutically acceptable cationic species such
as MgX or CaX, where X represents halo, hydroxyl, acetoxy,
trifluoroacetoxy, bisulfate, bicarbonate, or any other suitable species.
In some embodiments, M is a divalent cation, and is shared by two
molecules of formula (1).

[0127]The methods of the invention provide ways to synthesize the
oligosaccharide compounds within the invention. The methods include, for
example, ways to link two mannose units together in a desired fashion,
such as by forming a 1,6-alpha linkage between two mannose units. Where
the 1,6-alpha linkage is a phosphate or phosphonate such as the compounds
of formula (1) where X is O, the linkage can be created by a Mitsunobu
reaction. The Mitsunobu reaction is described, for example, in Campbell,
D. A., J. Org. Chem. (1992) 57:6331-6335, and involves an activation of
an alcohol with a reagent such as an azodicarboxylate, e.g., DEAD or
DIAD, and a phosphine (e.g., triphenyl phosphine or
tris(p-chlorophenyl)phosphine), followed by displacement of the activated
O by a nucleophilic species. In the case of compounds of formula (1)
where X is O, the nucleophile is usually a P--O.sup.- species, typically
a phosphonate or phosphate anion. In other aspects of this invention, a
Mitsunobu reaction is employed to introduce an aza substituent to a
saccharide group, in which case the conditions are similar except that
the nucleophile is an azide, which may be supplied as an azide anion or
as a trialkylsilyl azide or a phosphoryl azide.

[0128]The methods of the invention also provide ways to install and modify
N-substituents (aza substituent) on the mannose units of compounds such
as those of formula (1). Thus in some embodiments, the methods provide
ways to insert an aza substituent into a mannose unit with the proper
stereochemistry. One such method comprises displacing a C-2 hydroxyl on a
pyranose ring with an N-substituent, typically N3. It can be
inserted with a nucleophilic displacement reaction, but in some
embodiments it is inserted via a Mitsunobu-type reaction with the
assistance of an activating group such as an azodicarboxylate, e.g., DEAD
or DIAD. The nucleophile for this Mitsunobu reaction can be provided by a
phosphoryl azide such as diphenyl phosphoryl azide. The particular
improvement provided by the present invention in this reaction comprises
the use of a phosphoryl azide as the source of the aza substituent, and
the incorporation of substantially water-free work-up conditions to avoid
decomposition of the product. Under the conditions described herein, the
yield of the 2-azido derivative of a mannose unit is substantially
improved over methods of the prior art.

[0129]In some preferred embodiments, at least one of the mannose units of
an oligosaccharide of the invention is an aza-substituted mannose having
an amine or acylated amine, which is often at C-2 of the mannose unit. In
some embodiments of the methods of the invention, the aza substituent is
inserted as N3, which can be reduced to provide an amine, and the
amine can be acylated under conditions such as those described in the
Greene and Wuts book on protective groups referenced above. Reduction of
the azide (N3) to NH2 is readily accomplished by typical
methods, such as using nickel borohydride, which can be generated in situ
from nickel chloride hexahydrate and sodium borohydride as described
herein.

[0130]In some embodiments, the invention provides oligosaccharides and
methods to make oligosaccharides, wherein the oligosaccharide comprises a
repeating series of aza-substituted mannose units; in many embodiments,
each of the mannose units has an aza substituent, typically N3,
NH2 or NHAc, at C-2 of each mannose unit--e.g., in the compounds of
formula (1), Az would be N3, NH2 or NHAc. Optionally, the
linkage between at least two of the mannose units is a phosphonate
linkage which may be a 1,6-alpha linkage such as the one in the compounds
in formula (1), where W is CH2 and X is O. Such 1,6-alpha linkages
comprising a phosphate or a phosphonate, such as the compounds in formula
(1) where W is either CH2 or O and X is O, can be formed by the
Mitsunobu reaction described above. Preferably, the Mitsunobu is done
under modified conditions that are described by Campbell, as referenced
above.

[0131]In another aspect, the invention provides an improved method to make
certain saccharide molecules that are useful for synthesis of the
oligosaccharides described above. Thus the invention provides methods to
synthesize certain phosphonate-substituted monosaccharides. For example,
a compound of formula (2), which is available from glucose, can be
converted into a precursor for a phosphonate ester useful for forming the
phosphonate-comprising 1,6-alpha-linkage shown in formula (1) where W is
CH2 and X is O. The method includes cyclizing a compound of formula
(2) with an electrophile

##STR00016##

[0132]to form a compound of formula (3).

##STR00017##

[0133]wherein each of R2, R3, R4 and R6 is
independently H or a protecting group;

[0134]and El represents the residue of an electrophile.

[0135]The electrophile used to effect the cyclization may be a mercury
salt, in which case after cyclization to the compound of formula (3), the
residue of the electrophile is typically --HgX, where X is halo, acetoxy,
or the like. The mercury can be replaced by treatment with iodine, giving
a compound of formula (3) wherein El is I. The iodide can then be
displaced with a phosphorus nucleophile; for example, treatment with
trialkyl phosphite directly displaces the iodide and produces a dialkyl
phosphonate. Other electrophiles may also be used to effect the
cyclization; for example, halocyclizations are known, and would directly
provide a halide as El, which could be displaced. Similarly, methods can
be used whereby the olefin can be epoxidized with the correct
stereochemistry so that opening of the epoxide can result in the pyranose
ring, and a compound of formula (3), wherein El is OH. The OH can then be
converted to a halide or a displaceable sulfonate such as a triflate,
tosylate or mesylate, and the phosphorus can be introduced as described
above with triethyl phosphite, or it can be introduced by direct
nucleophilic substitution using an anionic P species such as the sodium
anion of diethyl phosphite.

[0136]This provides a glucose derivative of formula (3) rather than an
N-substituted mannose unit; thus the invention further provides methods
to convert OR2 in formula (2) or formula (3) into an aza substituent
having the desired `axial` orientation. This introduction of a nitrogen
substituent is often accomplished by reacting a compound of formula (3)
with an activating agent to form a compound wherein R2 is a
displaceable group, such as a triflate, tosylate or mesylate. Since such
displacements in (2) would result in allylic rearrangement, this
conversion is often done on a compound of formula (3). While it is
possible to do this reaction by ordinary nucleophilic displacements, in
one embodiment of the invention, this replacement of OR2 with an aza
substituent is done using a Mitsunobu reaction. In a preferred
embodiment, the Mitsunobu reaction employs a phosphoryl azide as the
source of the aza substituent. Thus, the reaction provides a very
efficient way to convert a readily available glucose-derived compound
such as (2) into a 2-deoxy-2-aza-substituted mannose derivative, wherein
the 2-aza-substituent is an azide. The azide can then be reduced to an
amine such as treatment with nickel borohydride; and the amine can be
functionalized or protected as desired. In many embodiments of the
invention, the amine is immediately acylated with a protecting group,
often with acetyl (Ac), since the acetyl group can serve as a protecting
group and also appears in the mannose unit of the CPS of N. meningitidis.

[0137]In another aspect, the invention provides a greatly improved method
for making a 2-aza-substituted mannose derivative in protected form. The
method comprises displacing the 2-hydroxyl group of an otherwise
protected glucopyranose ring by conversion of the 2-hydroxyl to a
triflate followed by displacement of the triflate with azide. The
nucleophilic displacement inverts the center, thus converting the
glucopyranose into a mannopyranose. While the displacement itself has
been reported, it proceeds in very poor isolated yield using standard
conditions. In the present invention, it has been found that a high yield
of the desired product can be obtained using the readily available
1,3,4,6-tetra-O-acyl glucopyranose, as long as the work-up of the
reaction is done substantially without water. Thus the triflate is
displaced by azide anion in a polar aprotic solvent such as DMF or NMP;
then, rather than using an ordinary aqueous/organic extractive workup to
remove most of the solvent and salts, the reaction mixture may be
partially concentrated before it is directly applied to a chromatography
column. Using such a non-aqueous work-up, the product can be eluted from
the column in high yield.

[0138]The following description provides more detail on the methods for
making certain compounds of the invention.

[0139]One embodiment of a starting monomer acceptor, compound XIV shown
below, is described in Berkin, A. et al., Chem. Eur. J. (2002)
8:4424-4433. As an alternative, the following sequence may be used:

##STR00018##

##STR00019##

[0140]When the di-benzyl protected compound was used to introduce the
spacer at the anomeric center, about a 1:1 mixture of anomers was
obtained. If instead the benzyl ethers are replaced with acetate esters,
only traces of the beta anomer are obtained, and an 86% isolated yield of
the desired alpha anomer resulted. Thus, the method provides an efficient
way to produce the desired alpha configuration at this center, which is
often the center to which a spacer connecting the oligosaccharide to a
carrier protein is attached. The alpha configuration is desired because
it best matches the all-alpha skeleton of the natural CPS of the target
organism. It was also found that the beta anomer was less reactive in the
ethylthio-glycoside replacement step. However, the acetates sometimes
cause difficulties later in the sequence, so in some embodiments, the
benzyl ethers were utilized for this step. The azido group of the fully
protected 2-aza mannose intermediate was reduced to an amine again using
NaBH4/cat. NiCl2-6H2O, and the amine was acetylated with
acetic anhydride in 85% yield over both steps. Finally, the silyl ether
was removed with TBAF to obtain the starting building block acceptor in
99% yield.

[0141]A convenient synthesis of the desired elongation monomers is shown
in Schemes 1-3 below, which allow one to prepare elongation monomers
having different protecting groups, for example.

##STR00020##

[0142]Compound I was obtained and used as a mixture of anomers, and was
obtained in seven steps from glucose. It was elongated via a Wittig
reaction with methyl bromotriphenylphosphine, for example, to afford the
alkene in 91% yield. Another preparation of this alkene from D-arabinose
used a two carbon elongation that involves addition of divinylzinc.
Mercury cyclization of compound II afforded exclusively the
alpha-C-glycoside, though the corresponding tetra-O-benzyl protected
alkene yields a mixture of anomers. Alternative methods for cyclizing
such species, such as haloetherification and epoxidation/electrophilic
ring opening may be used. The mercury acetate compound was converted to
the corresponding mercury chloride derivative, which was isolated. The
mercury chloride was subsequently replaced with iodine in excellent yield
by treatment with I2.

##STR00021##

[0143]To avoid formation of a cyclic phosphate in the next step, the C2
hydroxyl group was first protected as a silyl ether. The C-phosphonate
VII was obtained by treatment of the primary iodide V with triethyl
phosphite to give VI in 77% yield. The silyl ether was subsequently
cleaved from the C2 hydroxyl using tetrabutylammonium fluoride (TBAF) in
99% yield.

[0144]The axial acetamido group was introduced by a displacement reaction
with azide followed by reduction and acetylation as shown in Scheme 3.
The displacement inverts the C-2 center, providing the axial orientation
of the C-2 acetamide substituent that is found in the natural capsular
polysaccharide. This azide was introduced in low yield by preparing a
triflate of the C-2 hydroxyl and displacing it with azide anion. However,
by using Mitsunobu conditions and diphenylphosphoryl azide (DPPA) as the
azide source, a high yield (86%) of the azido derivative IX was obtained.
To introduce a selectively removable protecting group at O-6, the primary
benzyl ether of VIII was replaced with an acetate ester in 84% yield
using standard acetolysis conditions; that step could be omitted, in
which case all of the benzyl ethers could be cleaved at once. Omitting
the acetolysis shortens the synthesis, but makes it more difficult to use
the C-6 hydroxyl for further elongation or other functionalization,
although exchange of the benzyl for an acetyl can also be done after an
elongation step. The acetolysis reaction worked better when a mixture of
acetic anhydride and acetic acid (1/1) was used together with sulphuric
acid. The azide was reduced with sodium borohydride in the presence of
nickel chloride hexahydrate, and the resulting amine was acetylated with
acetic anhydride to give compound X in 69% yield.

[0145]Scheme 3 illustrates completion of an elongating monomer within the
invention that is a 2-aza mannose unit. For the construction of the
elongating C-phosphonate monomer a modified version of a published
approach was used. See Casero, F., et al., J. Org. Chem. (1996)
61:3428-3432. After desilylation of precursor VI to afford the 2-OH
compound VII, azide displacement using Mitsunobu conditions gave the
2-azido-2-deoxy-mannopyranoside VIII (86%). An orthogonal protecting
group, to allow later 6-O elongation, was introduced by acetolysis to
afford the 6-O-acetate IX (84%). Azide reduction followed by acetylation
gave X (76%), from which the ethyl esters were removed to yield the
elongating monomer XI with a free phosphonic acid. For many embodiments,
it is preferred to couple a mono-ester of the phosphonic acid, so that
the product remains protected as a phosphonate ester. These compounds can
be prepared in two steps as shown below for conversion of XI to XIII.
Note that the azide reduction can be postponed until additional mannose
units have been installed if longer oligosaccharides are desired, so that
all of the azides can be reduced and the resulting amines acetylated
simultaneously.

##STR00022##

[0146]The ethyl protecting groups on the phosphonic acid derivative were
replaced with methyls as shown in Scheme 3 above to facilitate removal
after an oligosaccharide has been constructed. Once the linked
disaccharide has been formed, the linkage is relatively labile under the
conditions needed to cleave the ethyl phosphonates. The ethyl groups were
thus removed quantitatively by treatment with bromotrimethyl silane
(TMSBr), and the phosphonic acid product was converted to its
corresponding dimethyl phosphonate in 84% yield using acetic acid and
trimethylorthoacetate. This reaction had to be carefully monitored since
deacetylation is a competing side reaction during prolonged reaction
times. Treatment of the dimethyl phosphonate with triethylamine (TEA) and
phenyl mercaptan efficiently afforded the mono methyl phosphonate XIII in
82% yield.

[0147]Scheme 4 illustrates the coupling reaction to establish a
phosphonate linkage between two mannose units. Both DCC and Mitsunobu
conditions have been reported for coupling a phosphonate to an alcohol
like the acceptor monomer, with yields in the range of 50-70%. Pozsgay,
et al. (See, A. Berkin, B. Coxon and V. Pozsgay, Chem. Eur. J, 2002, 8,
4424.) However, when the acceptor already contained a phosphonate, the
yields dropped sharply, which could limit the ability of such conditions
to provide the desired trimers, tetramers, and other longer
polysaccharides. Mitsunobu conditions in the present case, using
Ph3P and DIAD, gave a good yield (47%) of XV. However, substantially
better yields were realized using a modified version of the Mitsunobu
conditions, as described in Campbell, J. Org. Chem. (1992) 57:6331-6335.
The conditions from Campbell, using tris(4-chlorophenyl)phosphine and a
large excess of triethylamine, provided XV in an 88% yield. The best
yields are obtained in this reaction when care is taken to exclude other
nucleophilic species that could compete with the weakly-nucleophilic
phosphonate anion.

##STR00023##

[0148]Using the Mitsunobu conditions described above the yield in the
trimer formation was found to be comparable and even higher than in the
dimer formation. The dimer XV was transformed into a new acceptor
(→XVI) by deacetylation. The modified Mitsunobu reaction between
XVI and XIII then afforded the trimer XVII in a 92% yield. The identity
of the products was proven by MS and NMR, the latter partly complex due
to diastereomers phosphonate that result from the chirality of the
phosphorus. Compound XVII was deacetylated, which creates a new acceptor
XVIII which can be attached to another elongation monomer. After this and
subsequent removal of the phosphonate methyl ester, the chirality of the
phosphorus center was destroyed, and the NMR data for compound XIX
reflected the absence of phosphorus diastereomers.

##STR00024##

[0149]It was also found that by prolonging the exposure of the mixture of
the acceptor XIV and elongation monomer XIII to the Mitsunobu conditions,
and in the presence of some excess XIII, the reaction provides longer
monomers. Thus under the reaction conditions, cleavage of the C-6 acetyl
group from an incorporated elongation monomer occurs in situ at some
rate; and this provides a new acceptor molecule. Thus to some degree, for
example, conversion of XVI to XVI occurs by prolonged exposure to the
Mitsunobu reaction, and under those conditions in the presence of excess
XIII, it forms XVII; and so forth, to produce higher oligomers. This
reaction thus provides a way to make oligomers containing multiple
mannose units in protected form without requiring a stepwise
deprotection/isolation process, and allows the synthesis of immunogenic
oligosaccharides of the invention having a distribution of sizes. In some
embodiments, this has been shown to provide primarily tetra- and
penta-mannose oligosaccharides, with minor amounts of longer oligomers.
It thus provides a highly efficient way to synthesized longer oligomeric
compounds within the invention.

[0150]The invention also provides a simple and improved synthesis of
2-azido-2-deoxy-D-mannopyranose from a readily available precursor,
1,3,4,6-tetra-O-acetyl-glucopyranose, as illustrated in Scheme 5. This
intermediate is useful for the synthesis of many of the mannose units of
the present invention.

##STR00025##

[0151]In an attempt to synthesize a protected 2-azido mannose compound,
1,3,4,6-tetra-O-acetyl glucopyranose was converted into its 2-O-triflate
using triflic anhydride, and the triflate was treated with NaN3.
According to TLC and MALDI-TOF-ms, the 2-azido product forms in a high
yield, but apparently it substantially decomposes during a typical
aqueous workup, and the isolated yield was only 20%. However, when the
reaction was worked up without using water, the yield was sharply
improved. Thus most of the DMF used as the solvent for the displacement
was removed under reduced pressure, and the residue was transferred to a
dried column containing a slurry of pre-dried silica in dry toluene. The
product was eluted using a gradient of increasing ethyl acetate in
toluene. Once purified, the product was relatively stable. The
1,3,4,6-tetra-O-acetyl glucopyranose starting material is very easy to
synthesize in large scale in a one pot synthesis from glucose, and the
intermediate triflate was obtained using standard conditions in 97%
yield. The isolated yield from the inversion as mentioned is strongly
dependent on how water free the workup is: exclusion of water by using
pre-dried glassware and pre-dried silica as described above is necessary
for optimum yield. By minimizing exposure to moisture during work-up and
during silica gel chromatography, reproducible yields of 60% or better
were obtained.

[0152]The tetraacetate product shows some tendency to decompose during
storage. However, once converted to its 1-ethylthio glycoside by
treatment with EtSH/BF3-Et2O it was quite stable. It was next
treated with alkoxide to remove the acetate groups, as shown in Scheme 6,
and the primary hydroxyl was selectively protected with a
t-butyldimethylsilyl (TBS) group. The two secondary hydroxyls were
protected as benzyl esters, but a variety of different protecting groups
can be used here and in many other steps throughout this process. The
--SEt group was then replaced by a linker, which can be used to conjugate
the final oligosaccharide to a protein. Alternatively, it could be used
to attach the acceptor monomer to a solid support while the
oligosaccharide is assembled. In this case, a protected aminoethyl group
was introduced at the anomeric position; however, a wide variety of
linking groups that are suitable for conjugating the oligosaccharide may
be used, and those of skill in the art can readily select and use others.

##STR00026##

[0153]The dimer can be used as an immunogen without further elongation, or
longer oligomers can be prepared and used. For both the dimer and the
trimer, the acetyl group at C6 is removed by standard hydrolysis. The
methyl esters of the phosphonate were cleaved using
1,8-diaza-7-bicyclo[5.4.0]undecene (DBU) as the base along with phenyl
mercaptan, to effect a nucleophilic demethylation. The benzyl ethers were
then cleaved along with the benzyloxycarbonyl via a standard reductive
hydrogenation, using palladium on activated charcoal as the catalyst. To
get the reaction to go to completion, aqueous HCl was added so that the
resulting amine is protonated to form the hydrochloride, which reduces
its tendency to poison the catalyst.

[0154]The oligosaccharides of the invention can be administered to a
mammal to elicit an immunogenic response; they may be used as described
above, typically after at least partial deprotection. They may also be
partly or entirely acetylated to broaden the spectrum of the immune
response elicited. The CPS of the target organism is substantially
acetylated on the C-3 hydroxyl; thus the flexibility of the present
synthetic method makes it possible to prepare compounds containing one or
more acetyl groups, and particularly to prepare dimers, trimers, and
higher oligomers that are partially or entirely acetylated at C-3
hydroxyl, or mixtures of these.

[0155]Instead of, or in addition to, using phosphonate linkages to
stabilize the phosphodiester, as described above it is possible to
stabilize the phosphodiester to a useful degree by incorporating an azide
group as the C-2 aza substituent during synthetic manipulations up to and
optionally during the step of conjugating the oligosaccharide to a
protein. This stabilization is inductive, but is adequate to improve the
working stability of the intermediates over that of the corresponding
NHAc compounds. Once the azide has served its purpose, it can be reduced
with nickel borohydride and acetylated with acetic anhydride or similar
acylating agents as described herein. The oligomeric compounds of the
invention can thus incorporate phosphodiester linkages to some degree as
described above, and the phosphodiesters can be incorporated by typical
methods and those described herein.

[0156]To have the option to get both the target molecules with or without
acetates, the protecting group strategy was based on acetates as
permanent protecting groups. To stabilize the anomeric phosphodiester
linkage azide was used as a precursor for the acetamido group as far as
possible into the synthesis, as illustrated in Scheme 7 below. The
electron withdrawing properties and the lack of participating effect of
the azide strongly protects the linkage. And after initial problems with
the use of dimethoxy trityl as a temporary protecting group for the 6-OH
we used TBDMS (t-butyl dimethylsilyl ether, sometimes referred to as
TBS). The TBDMS ethers were cleaved off in good yield with minimal
problems due to acetyl migration or cleavage of the labile phosphodiester
linkage. TBAF which was successfully used for deprotection of TBDMS
ethers in the previous C-phosphonate synthesis could not be used due to
acetyl migration so the rather slow (especially when having azide in pos
2) but reliable if TREAT-HF was used.

##STR00027##

[0157]For an efficient modular synthesis, three monosaccharide building
blocks were needed; a spacer equipped starting monomer 6-OH acceptor, an
alpha-H-phosphonate elongating monomer with temporary 6-OH protection
(donor 1) and a terminating alpha-H-phosphonate monomer phosphorylated in
the primary position (donor 2). To achieve the target molecules with a
phosphate at C-6, as exemplified here as trimeric oligosaccharides, a
donor containing a dibenzyl protected phosphate was used because an
attempt at post-linkage modification of the C-6 hydroxyl using amidite
chemistry was unsuccessful.

[0158]To simplify the synthesis all three units were synthesized from the
same precursor, XXVIII, as shown in Scheme 7 above. The starting material
was synthesized using conventional methods. To make the acceptor, it was
coupled to a benzyloxycarbonyl protected ethanolamine spacer in 86% yield
and the TBDMS-ether removed using TREAT-HF to give the acceptor XXIX in
97% yield.

[0159]Donor 1 was synthesized by first hydrolyzing the thioglycoside using
a glycosylation with NIS as promoter and water as acceptor at -20°
C. The desired alpha-anomer was obtained exclusively in 82% yield. If the
reaction is carried out at room temperature, a large percentage of beta
is formed. This compound was then phosphonylated using
PCl3/imidazole giving XXX in 97% yield. Thus, the invention includes
preparing alpha-anomers at low temperatures such as lower than room
temperature, preferably lower than 0° C., more preferably lower
than -10° C., such as -20° C. or less.

[0160]To synthesize donor mannose units having the H-phosphonate, which is
useful to add mannose units linked to an oligosaccharide of the invention
through a phosphodiester linkage, the TBDMS-ether was cleaved in 75%
yield and the hydroxyl was phosphorylated using amidite chemistry to give
a benzyl protected phosphate at C-6 (67%). The ethyl thio glycoside was
hydrolyzed in the same fashion as for donor 1 by coupling to water (79%)
and then phosphonylated to give the H-phosphonate donor mannose unit XXXI
in 92% yield.

[0161]The coupling of the elongating monomer to the acceptor was carried
out using conventional H-phosphonate chemistry with a pivaloyl
chloride-promoted condensation as illustrated in Scheme 8 below. The
H-phosphonate diester was oxidized using elemental iodine and water to
provide the phosphate diester. The dimer was obtained in a good yield
(96%).

##STR00028##

[0162]Elongation to trisaccharides is achieved by deprotection of the C-6
hydroxyl with TREAT-HF and iteration of the addition of an elongation
monomer; and the compatibility of the protecting group strategy here with
that exemplified in the schemes above for the phosphonate linkage
construction enables the two types of linkages to be mixed or matched in
a given oligosaccharide. The example here illustrates elongation with the
donor molecules XXX and XXXI, providing a trimeric oligosaccharide having
either a C-6 hydroxyl on the last mannose unit, or a C-6 O-phosphate in
that position.

##STR00029##

[0163]To obtain the trisaccharide, the TBDMS-ether on the disaccharide was
removed in 91% yield, again using TREAT-HF. Subsequent coupling of the
two donors using the same conditions as employed for the coupling to the
disaccharide, resulted in the formation of the two trisaccharides in 62%
and 59% yield respectively. The yields obtained during the second or
subsequent couplings were lower than that obtained from the first
coupling step, as observed by Pozsgay, et al. (See, A. Berkin, B. Coxon
and V. Pozsgay, Chem. Eur. J., 2002, 8, 4424.) Thus in preferred
embodiments at least some of the linkages between adjacent mannose units
in an oligosaccharide typically comprise a phosphonate instead of a
phosphodiester.

[0164]The azides on both forms of the oligomers of the invention may also
comprise an additional phosphate in protected form at R' or its
equivalent position, e.g., the R6 group in dimers and trimers of the
phosphonate oligomers. This is optionally introduced because the terminal
mannose units in the natural CPS may be phosphorylated, and the
corresponding phosphoryl group can be introduced as a dibenzyl phosphate
ester using conventional chemistry. The benzyl groups can then be removed
along with the benzyl ethers at C-3 and/or C-4, for example, or along
with reductive cleavage of a benzyloxycarbonyl group that may be used to
protect an amine group in the spacer moiety at position 1 of the first
mannose unit. The benzyl phosphonate does not interfere with the other
reactions needed to produce the oligomers. For example, the azides in
certain compounds containing this group were successfully reduced to
amines using sodium borohydride and a catalytic amount of nickel chloride
hexahydrate, and the resulting amines were acetylated with acetic
anhydride giving the corresponding aminoacyl derivatives. This reaction
proceeded in 89% yield when the terminal group on a mannose unit in a
trimeric phosphodiester-linked compound was OTBDMS, and in 64% yield when
that terminal group was a dibenzyl phosphate.

##STR00030##

[0165]The two trisaccharides were deprotected in a different order. To
achieve the target molecules 1 and 2 first the Z-group was deprotected
using reductive hydrogenation (83%). To avoid problems with degradation
of the phosphodiester linkage during reductive hydrogenation, basic ion
exchange resin were added to the mixture with good result. The
TBDMS-ether was removed again using TREAT-HF giving 1 in 85% yield. The
acetate was cleaved under standard sodium methoxide/methanol conditions.
For target molecule 3 the Z was removed in the same way as in the
previous deprotection of 1 (85%) and to achieve 4 the acetates were
removed (80%) prior to the reductive hydrogenation (64%).

[0166]The methods described in the schemes above may also be used to make
compounds having a C-phosphonate, such as those compounds of formula (1)
or (1'), described herein.

[0167]Use of the Compounds as Antigens

[0168]The oligosaccharides prepared by the above methods are immunogenic
and are useful to elicit an immune response that protects against
infection by N. meningitidis, as in a vaccine. In particular, an
oligosaccharide having the structure shown in formula (1) above is
capable of eliciting an immunogenic response if administered to a mammal,
and therefore administering composition comprising an oligosaccharide of
formula (1) is useful to elicit the formation of antibodies and/or to
provide an immunogenic response and to provide at least partial
resistance or partial immunity to infection by N. meningitidis A. The
antibodies and/or immunogenic response provide protection against
infection by N. meningitidis A. Compounds comprising the moiety in
formula (1), especially those wherein W is CH2 or O, X is O, and Az
is NHAc, are useful as vaccine components, as are compounds of formula
(4).

[0169]These compounds are especially useful when conjugated to a protein,
and optionally the Z group of formula (1) may be used to link the
oligosaccharide of formula (1) to a protein. Likewise, in other compounds
of the formula, the corresponding group at C-1 of the first mannose unit
in an oligosaccharide of the invention may be a moiety capable of being
used to conjugate the compound to a carrier protein to improve its
immunogenic properties. Notwithstanding the option of linking through Z,
however, the compounds of the invention and specifically compounds
comprising the structure in formula (1) or formula (4) can be linked by
other means to a carrier protein, such as through one or more of the
hydroxyl groups or through an acyl group attached in place of an acetyl
group on one of the acetylamines on one of the mannose units. Such
alternative conjugation mechanisms are within the scope of the invention,
as long as the C-1 (anomeric) center of the oligosaccharide remains in
the desired alpha configuration. Preferably such oligosaccharides
comprise at least one phosphonate linkage between two mannose units.

[0170]Accordingly, an aspect the present invention is directed to vaccines
which contain as an active ingredient an immunogenically effective amount
of an immunogenic oligosaccharide as described herein. The
oligosaccharide(s) may be introduced into a host, including humans,
either alone or linked to a carrier such as a protein or as a homopolymer
or heteropolymer of mannose units or of other saccharides. In some
embodiments, they are used as glycoconjugates, and the protein to which
the oligosaccharide is conjugated is selected for its ability to enhance
the immunogenic response to the oligosaccharide antigen: proteins that
have elicited an immune response in the mammal to be treated are
sometimes preferred. The glycoconjugates have the advantage of increased
immunological activity and the additional ability to induce antibodies
and/or CTLs that react with different antigenic determinants of the virus
or tumor cells.

[0171]The immunogenic compositions of the invention include at least one
compound of the invention, and may optionally include more than one such
compound. Thus it is sometimes desirable to mix together two or more
compounds of the invention to broaden the immunogenic response elicited.
For example, it is often desirable to combine a compound wherein R3
is H with a compound wherein R3 is Ac, since the CPS of the target
organism is partially, but not fully, acetylated at this position. Thus
in some embodiments, the immunogenic compositions comprise at least two
and optionally three or more of such immunogenic compounds and/or protein
conjugates of such compounds.

[0172]Similarly, the carrier proteins may influence the immunogenic
response and even affect the precise nature of the antibodies that result
from treatment of a mammal with one or more compounds of the invention
when delivered as glycoconjugates. Therefore, the invention includes
compositions that comprise more than one carrier protein, whether or not
they comprise two or more oligosaccharides.

[0174]Carrier proteins should be amenable to standard conjugation
procedures. In a preferred embodiment of the present invention diphtheria
toxin purified from cultures of Corynebacteria diphtheriae and chemically
detoxified using formaldehyde is used as the carrier protein. In other
embodiments, CRM197 was used.

[0175]Methods for attaching an oligosaccharide to a carrier protein are
conventional, and a skilled practitioner can create glycoconjugates
comprising the compounds of the invention using conventional methods.
Guidance is also available in many publications and in, for example, U.S.
Pat. Nos. 4,356,170; 4,619,828; 5,153,312; 5,422,427; and 5,445,817.

[0176]In one such embodiment, using 2-aminoethoxy as the spacer moiety at
C-1 of the first mannose unit and a phosphodiester linkage, and using
either suberic acid or squarate as the bi-functional reagent to connect
the spacer moiety to the carrier protein, and using CRM197 as the carrier
protein, glycoconjugates were prepared. According to MALDI-TOF mass
spectral analysis, these conjugates contained about five oligosaccharide
molecules, on average, per carrier protein molecule. However,
oligosaccharide:carrier protein ratios of about 0.1 to about one on a
molar ratio are often desirable, and molar ratios of about 1-30:1 or
about 1-10:1 are within the scope of the invention, regardless of which
carrier protein is utilized. The ratio of oligosaccharide to carrier
protein may also be characterized as a weight ratio between the two;
ratios of about 1:1 to 1:500 are often preferred. The precise ratio
depends upon the ratio of the molecular weight of the protein and the
oligosaccharide; for smaller oligosaccharides, a weight ratio of 1:5 to
1:200 is sometimes used.

[0177]After conjugation of the capsular polysaccharide to the carrier
protein, the polysaccharide-protein conjugates may be purified (enriched
with respect to the amount of polysaccharide-protein conjugate) by a
variety of techniques. One goal of the purification step is to remove the
unbound polysaccharide from the polysaccharide-protein conjugate. One
method for purification, involving ultrafiltration in the presence of
ammonium sulfate, is described in U.S. Pat. No. 6,146,902. Alternatively,
conjugates can be purified away from unreacted protein and polysaccharide
by any number of standard techniques including, inter alia, size
exclusion chromatography, density gradient centrifugation, hydrophobic
interaction chromatography or ammonium sulfate fractionation. See, e.g.,
Anderson, P. W., et al., J. Immunol. (1986) 137:1181-1186. See also
Jennings, H. J., et al., J. Immunol. (1981) 127:1011-1018.

[0178]The vaccines of the invention comprise at least one immunogenic
compound of the invention, and can also contain a physiologically
tolerable (acceptable) diluent such as water, phosphate buffered saline,
or saline, and further typically include an adjuvant. U.S. Pat. No.
6,869,607 provides non-ionic adjuvants for protein-conjugated immunogens
and discusses other suitable adjuvants, and is incorporated by reference
for its description of such adjuvants. Materials such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum,
aluminum sulfate, aluminum phosphate, and combinations thereof are
materials well known in the art as adjuvants. Also useful are BAY,
DC-chol, pcpp, monophosphoryl lipid A, CpG, QS-21, cholera toxin, and
formyl methionyl peptide, as well as emulsion formulations including
MF59, SAF, squalane, Tween, pluronic-blocked polymer L121, and components
such as monophosphorylipid A, trehalose dimycolate, and cell wall
skeleton. Cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6. IL-7, IL-12,
interferons, and tumor necrosis factor (TNF) may also be included. In
some preferred embodiments of the invention, alum is used as the
adjuvant.

[0179]Upon immunization with a composition as described herein, via
injection, aerosol, oral, transdermal or other route, the immune system
of the host responds to the vaccine by producing large amounts of CTLs
specific for the desired antigen, and the host becomes at least partially
immune to later infection by the N. meningitidis pathogen.

[0180]The vaccines and pharmaceutical compositions of the invention are
intended for parenteral, topical, oral or local administration.
Preferably, they are administered parenterally, e.g., intravenously,
subcutaneously, intradermally, or intramuscularly. Thus, the invention
provides compositions for parenteral administration which comprise a
solution of the immunogenic moiety dissolved or suspended in an
acceptable carrier, preferably a primarily aqueous carrier. A variety of
aqueous carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine, hyaluronic acid and the like. These compositions may be
sterilized by conventional sterilization techniques, or may be sterile
filtered. The resulting aqueous solutions may be packaged for use as is,
or lyophilized, the lyophilized preparation being combined with a sterile
solution prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate,
etc. The preparation of such pharmaceutical compositions is within the
ordinary skill in the art, and may be guided by standard reference books
such as Remington's Pharmaceutical Science, latest ed., which is
incorporated herein by reference.

[0181]In preferred embodiments, a vaccine composition comprising at least
one moiety of formula (1) or formula (4) in a substantially aqueous
mixture is formulated as a sterile, pyrogen-free buffered saline or
phosphate-containing solution, which may include a preservative or may be
preservative free. Suitable preservatives include benzyl alcohol,
parabens, thimerosal, chlorobutanol, and benzalkonium chloride, for
example. The solution is preferably approximately isotonic, and its
tonicity may be adjusted with agents such as sodium tartrate, sodium
chloride, propylene glycol, and sodium phosphate.

[0182]The concentration of the immunogenic oligosaccharides of the
invention in the pharmaceutical formulations can vary widely, i.e., from
less than about 0.1%, usually at or at least about 0.1% to as much as 20%
to 50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., and in accordance with the particular mode of
administration selected. A human unit dose form of the compounds and
composition is typically included in a pharmaceutical composition that
comprises a human unit dose of an acceptable carrier, preferably an
aqueous carrier, and is administered in a volume of fluid that is known
by those of skill in the art to be used for administration of such
compositions to humans, and is adjusted according to commonly understood
principles for a particular subject to be treated. Thus in one
embodiment, the invention provides a unit dosage of the vaccine
components of the invention in a suitable amount of an aqueous solution,
such as 0.1-3 ml, preferably 0.2-2 mL.

[0183]The immunogens of the invention may also be administered via
liposomes, which can serve to concentrate them in a particular tissue,
such as lymphoid tissue, or to enhance their activity or delivery
characteristics. A variety of methods are available for preparing
liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys.
Bioeng. (1980) 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369.

[0184]For solid compositions, conventional nontoxic solid carriers may be
used which include, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like. For oral
administration, a pharmaceutically acceptable nontoxic composition is
formed by incorporating any of the normally employed excipients, such as
those carriers previously listed, and a unit dosage of an active
ingredient, that is, one or more compounds of the invention, whether
conjugated to a protein or not.

[0185]For aerosol administration, the immunogenic compounds are preferably
supplied in finely divided form along with a surfactant and propellant.
The surfactant must, of course, be nontoxic, and preferably soluble in
the propellant. Representative of such agents are the esters or partial
esters of fatty acids containing from 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids with an aliphatic polyhydric alcohol or its
cyclic anhydride. Mixed esters, such as mixed or natural glycerides may
be employed. The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be included, as desired, as
with, e.g., lecithin for intranasal delivery.

[0186]Amounts effective for this use will depend on, e.g., the
oligosaccharide composition, whether it is conjugated to a protein, the
manner of administration, the weight and general state of health of the
patient, and the judgment of the prescribing physician, but generally
range for the initial immunization (that is for a prophylactic
administration) from about 1.0 μg to about 5,000 μg of peptide for
a 70 kg patient, (e.g., 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg, 3.0
μg, 3.5 μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.5 μg, 10 μg,
12.5 μg, 15 μg, 17.5 μg, 20 μg, 25 μg, 30 μg, 35 μg,
40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 250 μg, 500 μg,
750 μg, 1,000 μg, 1,500 μg, 2,000 μg, 2,500 μg, 3,000
μg, 3,500 μg, 4,000 μg, 4,500 μg or 5,000 μg). The actual
dose administered to a subject is often determined according to an
appropriate amount per kg of the subject's body weight. For example, an
effective amount may be about 0.1 to 5 μg/kg body weight.

[0188]The immunogenic compositions comprising a compound of the invention
are suitable for use in adult humans or in children, and in some
embodiments they are administered to young children, who are at risk for
contracting meningitis caused by N. meningitidis. Optionally such a
composition may be administered in combination with other
pharmaceutically active substances, and frequently it will be
administered in combination with other vaccines as part of a childhood
vaccination program. Compositions for administration may beneficially
include other types of immunogenic compounds such as glycoconjugates that
elicit an immune response to provide protection against other meningitis
pathogens, also. Thus in some embodiments, the vaccine compositions of
the invention include at least one antigen derived from another
Meningitidis serotype, typically from at least one of serotypes A, B, C,
W135, and Y. Preferred embodiments often comprise antigens to at least
one and often two or all of serotypes C, Y and W135 in combination with
at least one and optionally two or more compounds comprising an
oligosaccharide of the present invention.

EXAMPLES

[0189]The following examples illustrate certain embodiments of the
invention, but in no way limit its scope. Other variations of the
exemplified reactions and formulations will be apparent to those skilled
in the art, and are also within the scope of the invention. For example,
changing the order of steps, or modifying the protecting group strategy
selected often represent typical choices made by the ordinary
practitioner. The numbering of compounds in the following section does
not correlate to the numbering of compounds in the reaction schemes
provided above, and is provided for the convenience of the reader.

[0197]Dimethyl
C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-mannopyranosy-
l) methanephosphonate (7). To a solution of 5 (0.25 g, 0.43 mmol) in dry
CH2Cl2 (5 mL) bromotrimethylsilane (0.28 mL, 2.2 mmol, 5 eq)
was added at rt. After the addition was complete, the mixture was stirred
at rt for 1 h. The solution was cooled to 0° C. and Et3N (1
mL) was added followed by addition of water (1 mL). After 10 min the
solvents were removed under reduced pressure and the residue desalted by
RP-chromatography (water-water/MeOH 1:1). The product
C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-mannopyranosy-
l)methanephosphonate (6) was methylated without further purification. To
the product from the reaction described above were added AcOH (5.5 mL)
and trimethylorthoacetate (11.5 mL). The mixture was heated at reflux for
30 min and then concentrated. The residue was purified by silica gel
chromatography to give 7 (0.20 g, 0.36 mmol, 84% over two steps). (The
reaction has to be monitored carefully and stopped exactly when the
reaction is completed, otherwise the result is a mixture of the wanted
product and the product where the 6-OAc is deprotected.) 13C-NMR
(CDCl3) 170.6, 170.0, 137.4, 137.1 remaining aromatic C 129.9-127.4,
75.3, 73.0, 72.5, 72.4, 72.0, 66.7, 61.8, 52.9, 52.8, 52.2, 52.1, 49.0,
48.7, 28.0 (d, J=1142 Hz), 23.2, 20.8; 31P-NMR (CDCl3) 31.6.

[0201]2-Carboxybenzylamidoethyl 6-O-[methyl
C-(2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-mannopyranosyl)methaneph-
osphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-mannopyranoside
(11). To a solution of 10 (73 mg, 0.066 mmol) in MeOH (2 mL), KOH (7.4
mg, 0.13 mmol) from a stock solution of KOH dissolved in MeOH were added.
After 25 min the reaction mixture was diluted with CH2Cl2,
filtered through a plug of silica and the solvents were removed under
reduced pressure. The residue was purified on a LH-20 gel column
(CH2Cl2/MeOH 4:1) to give 11 (70 mg 0.066 mmol, 100%).

[0232]To study the influence of C-phosphonate in place of phosphodiester
on the immunogenicity and stability of the MenA saccharide, a synthetic
oligosaccharide analogous to trimers of the repeating unit of Neisseria
meningitidis group A polysaccharide was synthesized and conjugated to the
carrier protein CRM197. The glycoconjugate was analyzed in order to
verify physicochemical characteristics and immunogenicity in mice. The
structure of the conjugate is below.

##STR00039##

[0233]This compound was obtained by replacing the phosphodiester groups
linking the monosaccharides, with a C-phosphonate group in the
non-acetylated and non-phosphorylated base molecule.

[0237]The freeze dried product as reconstituted with saline (final
concentration=0.8 mg/ml) and assayed by calorimetric test for total
phosphorus and protein content (MicroBCA assay). The phosphorus content
was then converted to total saccharide content using the appropriate
converting factor.

[0238]Because some precipitate was generated during storage of the
conjugate, a concentrate phosphate buffer (Na phosphate 100 mM pH 7.5)
was added to reach 10 mM as final concentration. Then the concentration
was recalculated considering the dilution factor. The conjugates had a
total saccharide content of 97.10 μg/ml and protein content of 215.63
μg/ml after dilution with phosphate buffer.

[0239]b) Immunogenicity in Mice

[0240]The immunogenicity of the conjugate with the synthetic
oligosaccharide was analyzed in ELISA and bactericidal assays. BALB/c
mice (8/group) were immunized with the purified glycoconjugates
formulated with and without alum phosphate as adjuvant. The immunisation
protocol, shown in Table 1, included three doses (200 ng each) at days 0,
14 and 28 with a final bleeding at day 42. Post 1, post 2 and post 3 dose
sera were analyzed to determine specific total IgG and the corresponding
antibody functional activity. MenA-CRM 2011 is the wild type MenA
conjugate.

[0242]Sera were analyzed in ELISA assay for specific determination of IgG
antibody responses in mice sera. Total IgG titres against A, C, W135 and
Y polysaccharide antigens (MenA, MenC, MenW, MenY) were calculated by
linear regression curves and expressed as ELISA units/ml (EU/ml). Sera
titres resulted from the comparison to the titration curve of a standard
serum.

[0243]Microtitre plates were coated with a mixture of capsular
polysaccharide and methylated human serum albumin at a final
concentration of 5.0 μg/ml each, in PBS pH 7.4. Plates were sealed,
incubated overnight at 2°-8° C., then washed and saturated
with a buffer containing 5% fetal calf serum, as blocking reagent, in PBS
pH 7.4. After one hour incubation at room temperature, plates were washed
and diluted test sera were added to wells in row 1. Sera were analyzed by
a titration curve with a two-fold dilution step. After overnight
incubation at 2°-8° C., plates were washed and a goat
anti-mouse antibody conjugated to alkaline phosphatase was diluted 1:2000
in saturation buffer and added to the plates. The secondary antibody was
incubated for 2 hours at 37° C. and, after washing, plates were
added with chromogenic substrate solution (1 mg/ml p-nitro phenyl
phosphate in 1 M diethanolamine buffer pH 9.8, 0.5 mM MgCl2, 0.02%
NaN3). Reactions were incubated for 30 min at room temperature and
absorbance values were read at 405-620 nm wavelength.

[0244]Sera with absorbance values of first point superior to that of the
standard were retested using a higher initial dilution.

[0245]Mice with negative antibody response (O.D. values of first point
lower than 0.300 at dilution 1:100) were assigned a titre of 2 EU/ml and
classified as "non-responders".

[0248]The assay protocol was based on the inoculum of the test strain in
Mueller Hinton broth (Difco) with the addition of 0.25% glucose, starting
from isolated colonies grown on agar chocolate. Bacterial culture was
incubated at 37° C. with 5% CO2 and the growth stopped when
bacteria reached early exponential phase (O.D.600 0.220-0.240). The
culture was diluted to 10-4 Colony Forming Units (CFU)/ml in Gey's
balanced salt solution with 1% BSA and incubated for 1 hour at 37°
C. with 5% CO2, in the presence of heat inactivated test sera pools
and baby rabbit complement. Before (T0) and one hour after (T1) the
incubation, reaction mixtures were plated onto Mueller Hinton agar
(Difco). Plates were incubated overnight at 37° C. with 5%
CO2 and CFU/ml corresponding to T0 and T1 were counted.

[0252]The results of physicochemical analyses indicate that the
glycosylation degree of the conjugate is very low. The low conjugation
degree is indicated also by the modest signals obtained in Western Blot
and by the small differences noticed in SDS-Page between the conjugates
and the CRM reference. The conjugation conditions as well as the
purification of the conjugates may be optimized.

[0253]ELISA results

[0254]Results obtained are reported in Table 2 and FIG. 4. ELISA titres
from each single animal were measured and are expressed in ELISA Unit/ml
(EU/ml). The specific antibody response to each glycoconjugate was
calculated as the Geometric Mean Titre (GMT) of the corresponding
immunisation group.

[0255]Post 2 dose sera showed low titre in the synthetic conjugate
formulated with the adjuvant and any titre in immunization groups treated
without the adjuvant.

[0256]The conjugate did not show any titre with and without alum.

[0257]As expected, control groups treated with and without the adjuvant
showed high responses.

[0259]Serum bactericidal titres were determined in sera pools prepared
from each immunization group. The bactericidal titre is expressed as the
reciprocal serum dilution resulting in 50% decrease in CFU/ml of reaction
mixtures compared to the control CFU/ml measured at T0. Bactericidal
titres were measured against the serogroup A strain F8238. SBAb antibody
titres are reported in Table 3. (MenA-CRM is the wild type control.)

[0262]In control groups, titres were high in response to immunizations
either with or without the adjuvant.

[0263]In post 3 sera pools, titres increased compared to post 2 dose sera.
In groups treated with the synthetic conjugate without alum titres were
not measurable in immunizations with the adjuvanted conjugate titres were
measurable but lower.

[0264]Titres of control immunizations performed with the oligoconjugate
were significantly higher.

[0265]The results give evidence that the synthetic MenA oligosaccharide
conjugated to the protein carrier CRM197 is immunogenic in mice. The
conjugates have C-phosphonate linkages that should be much more stable
than the native phosphodiester bonds. The fact that some immunogenicity
has been detected is promising.

[0266]It will be appreciated that the foregoing examples are provided to
illustrate the invention, not to limit its scope. Various variations and
combinations of the elements disclosed are viable as one of ordinary
skill would recognize, and these are also within the scope of the
invention. For example, a wide array of protecting groups for amine and
hydroxyl groups are well known, and many of these can be used in place of
the ones specifically recited herein without departing from the spirit of
the invention.